Bioproduction of chemicals

ABSTRACT

The present invention provides various combinations of genetic modifications to a transformed host cell that provide increase conversion of carbon to a chemical product. The present invention also provides methods of fermentation and methods of making various chemical products.

BACKGROUND OF THE INVENTION

There is a need for alternative production methods of industrialchemicals used for various consumer products and fuels that arecurrently made from petroleum. One alternative method is the use ofengineered microorganisms to produce industrial chemicals. Currently, inthe field of bioproduced chemicals there is a need to improve microbialenzyme performance, enhanced production rate in order to reach the goalof becoming an at-cost replacement basis for petro-based chemicals.

A common challenge faced in field of bio-produced chemicals inmicroorganisms is that any one modification to a host cell may requirecoordination with other modifications in order to successfully enhancechemical bioproduction.

The current invention provides methods, systems of fermentation,genetically modified microorganisms, modified enhanced enzymes forchemical production, all of which may be used in various combinations toincrease chemical production of a desired chemical product.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

The present invention relates to genetically modified organisms capableof producing an industrial chemical product of interest, wherein thegenetic modification includes introduction of nucleic acid sequencescoding for polynucleotides encoding one or more of the following: (1) anacetyl-CoA carboxylase gene with one or more of its subunits fusedtogether in the genetic structure of the organism; (2) an acetyl-CoAcarboxylase gene having a predefined stoichiometric ratio of each of thefour ACCase subunits relative to one another; (3) a monofunctionalmalonyl-CoA reductase gene capable of catalyzing the conversion ofmalonyl-CoA to malonate semialdehyde and one or more genes encoding oneor more of the following enzymes: ydfG, mmsB, NDSD, rutE, and nemA; (4)a monofunctional malonyl-CoA reductase gene capable of catalyzing theconversion of malonyl-CoA to malonate semialdehyde and one or more genesencoding one or more enzymes capable of converting malonate semialdehydeketo form to 3-HP, and one or more genes encoding one or more enzymescapable of converting either the malonate semialdehyde enol form or themalonate semialdehyde hydrated form to 3-HP; (5) a monofunctionalmalonyl-CoA reductase enzyme fused to a dehydrogenase enzyme that iseither: (a) primarily not NADPH-dependent, (b) primarily NADH-dependent,(c) primarily flavin-dependent, (d) less susceptible to 3-HP inhibitionat high concentration, and/or (e) catalyzes a reaction pathway to 3-HPthat is substantially irreversible; (6) a monofunctional malonyl-CoAreductase enzyme fused to one or more malonate semialdehydedehydrogenase enzymes; (7) a malonyl-CoA reductase gene that is mutatedto enhance its activity at lower temperatures; (8) salt-tolerantenzymes; (9) a gene that facilitates the exportation of a chemicalproduct of interest or the export of an inhibitory chemical from withinthe cell to the extracellular media; and/or (10) a gene that facilitatesthe importation from the extracellular media to within the cell of areactant, precursor, and/or metabolite used in the organism's productionpathway for producing a chemical product of interest.

The present invention further relates to methods of producing a chemicalproduct using the genetically modified organisms of the invention. Thepresent invention further includes products made from these methods. Inaccordance with certain embodiments that product is acetyl-CoA,malonyl-CoA, malonate semialdehyde, 3-hydroxypropionic acid (3-HP),acrylic acid, 1,3 propanediol, malonic acid, ethyl 3-HP, propiolactone,acrylonitrile, acrylamide, methyl acrylate, a polymer including superabsorbent polymers and polyacrylic acid, or a consumer product.

The present invention further relates to a method of producing achemical product from a renewable carbon source through a bioproductionprocess that comprises a controlled multi-phase production processwherein the initiation and/or completion of one or more phases of theproduction process is controlled by genetic modifications to theorganism producing the chemical product and/or is controlled by changesmade to the cell environment. In accordance with this aspect of theinvention, the bioproduction process may include two or more of thefollowing phases: (1) growth phase; (2) induction phase; and (3)production phase. The present invention further includes products madefrom these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 Depicts some embodiments of the metabolic pathways to produce3-hydroxypropionic acid.

FIG. 2 Depicts some embodiments of the of various equilibrium states inthe malonate semialdehyde to 3-HP reaction in a cell environment.

FIG. 3 Depicts some embodiments of the reaction catalyzed by acetyl-CoAcarboxylase (ACCase)

FIG. 4 Shows the inhibition of ACCase enzyme activity by high saltconcentration

FIG. 5 Depicts some embodiments of the fusion ACCase subunit geneconstructs overexpressed in E. coli.

FIG. 6 Show improved production of 3-HP by genetically modified organismwith DA fusion ACCase

FIG. 7 Shows improved production of 3-HP by genetically modifiedorganism with overexpression of rhtA exporter.

FIG. 8 Shows various embodiments of the genetic modules used foroptimizing expression in host cells.

FIG. 9 Shows various chemical products that can made from variousembodiments of the invention.

Table 1 Lists the accession numbers for genes encoding ACCase subunitsfrom Halomonas elongate.

Table 2 Depicts some embodiments of the RBS sequences used to enhanceexpression of H. elongate ACCase subunits.

Table 3 Shows the improvement in 3-HP production by RBS-optimizedexpression of H. elongata ACCase subunits.

Table 4 Shows some embodiments of the ACCase subunit fusions thatincrease and ACCase enzyme complex activity.

Table 5 Shows some of the genetic modifications of a host cell forincrease chemical production.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “homology” refers to the optimal alignment of sequences (eithernucleotides or amino acids), which may be conducted by computerizedimplementations of algorithms. “Homology”, with regard topolynucleotides, for example, may be determined by analysis with BLASTNversion 2.0 using the default parameters. “Homology”, with respect topolypeptides (i.e., amino acids), may be determined using a program,such as BLASTP version 2.2.2 with the default parameters, which alignsthe polypeptide or fragments (and can also align nucleotide fragments)being compared and determines the extent of amino acid identity orsimilarity between them. It will be appreciated that amino acid“homology” includes conservative substitutions, i.e. those thatsubstitute a given amino acid in a polypeptide by another amino acid ofsimilar characteristics. Typically seen as conservative substitutionsare the following replacements: replacements of an aliphatic amino acidsuch as Ala, VaI, Leu and He with another aliphatic amino acid;replacement of a Ser with a Thr or vice versa; replacement of an acidicresidue such as Asp or GIu with another acidic residue; replacement of aresidue bearing an amide group, such as Asn or GIn, with another residuebearing an amide group; exchange of a basic residue such as Lys or Argwith another basic residue; and replacement of an aromatic residue suchas Phe or Tyr with another aromatic residue. For example, homologs canhave at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%,89%, 88%,87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid ornucleotide identity to the gene or proteins of the invention; or canhave 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%,89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81% or 80% amino acid or nucleotide to theessential protein functional domains of the gene or proteins of theinvention; or at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%,89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid ornucleotide to the essential binding amino acids within an essentialfunctional domain of the gene or proteins of the invention.

The above descriptions and methods for sequence homology are intended tobe exemplary and it is recognized that this concept is well-understoodin the art. Further, it is appreciated that nucleic acid sequences maybe varied and still provide a functional enzyme, and such variations arewithin the scope of the present invention. The term “enzyme homolog” canalso mean a functional variant.

The term “Functional homolog” means a polypeptide that is determined topossess an enzymatic activity and specificity of an enzyme of interestbut which has an amino acid sequence different from such enzyme ofinterest. A corresponding “homolog nucleic acid sequence” may beconstructed that is determined to encode such an identified enzymaticfunctional variant.

The term “3-HP” means 3-hydroxypropionic acid.

The term “heterologous DNA,” “heterologous nucleic acid sequence,” andthe like as used herein refers to a nucleic acid sequence wherein atleast one of the following is true: (a) the sequence of nucleic acids isforeign to (i.e., not naturally found in) a given host microorganism;(b) the sequence may be naturally found in a given host microorganism,but in an unnatural (e.g., greater than expected) amount; or (c) thesequence of nucleic acids comprises two or more subsequences that arenot found in the same relationship to each other in nature. For example,regarding instance (c), a heterologous nucleic acid sequence that isrecombinantly produced will have two or more sequences from unrelatedgenes arranged to make a new functional nucleic acid. Embodiments of thepresent invention may result from introduction of an expression vectorinto a host microorganism, wherein the expression vector contains anucleic acid sequence coding for an enzyme that is, or is not, normallyfound in a host microorganism. With reference to the hostmicroorganism's genome prior to the introduction of the heterologousnucleic acid sequence, then, the nucleic acid sequence that codes forthe enzyme is heterologous (whether or not the heterologous nucleic acidsequence is introduced into that genome). The term “heterologous” isintended to include the term “exogenous” as the latter term is generallyused in the art as well as “endogenous”.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “microorganism” includes a single microorganismas well as a plurality of microorganisms; and the like.

-   -   I. INTRODUCTION

The present invention relates to various genetically modifiedmicroorganisms, methods for making the same, and use of the same inmaking industrial products. Any and all of the microorganisms herein mayinclude a combination of genetic alterations as described herein. Thepresent invention contemplates, for example, a genetically modifiedmicroorganism having one or more of the following genetic modifications(i) an alteration that affects the stoichiometric ratio, expression orproduction of one or more ACCase enzyme genes (ii) a recombinant ACCasegene having at least 80% sequence homology to an ACCase gene from a salttolerant organism (iii) a genetic alteration in one or more non-ACCasegenes (iv) one or more genetic alterations that encodes for one or moreexporters capable of exporting 3-HP out of a cell (v) new hybridmolecules or co-expressed of a mono-functional malonyl-CoA reductaseenzyme with various 3-HP dehydrogenase proteins that: (a) exhibit lessinhibition by high 3-HP concentrations (b) that is less reversible orirreversible (c) enzymes that utilized NADH (d) enzymes that utilizedflavin (vi) one or more genetic alterations that can be used to switchthe carbon in the standard metabolic pathways of the cells to a pathwayengineered to produce a chemical. More details about each of the abovemodifications and how the modification are used together to increasechemical production in a host cell is described below.

The present invention also relates to methods of fermentation. Thegenetically modified microorganisms are cultured under conditions thatoptimized a host cell for increase chemical production. Thebio-production process may include two or more of the following phasesof fermentation: (1) growth phase where the culture organism replicatesitself and the carbon intermediate product is built up; (2) theinduction phase, where the expression of key enzymes critical to thechemical production is induced and the enzymes accumulate within theorganism to carry out the engineered pathway reactions required tofurther produce the chemical product (3) production phase is where theorganism expresses proteins that provide for continuously production thedesired chemical product. The above phases are further controlled by (1)addition and amount of the initiating reactant added to the reactionvessel (2) key enzymes engineered into the organism using promoters thatare sensitive to (e.g., activated by) the depletion of the initiatingreactant. Addition details about the fermentation process of theinvention are disclosed below.

-   -   II. ACETYL-CoA CARBOXYLASE        -   Malonyl-CoA Flux

One of the steps in the biosynthesis of 3-HP involves the reactioncatalyzed by acetyl-CoA carboxylase (ACCase) enzyme. ACCase is a primarycontrol point in the 3-HP pathway shown in FIG. 1 (previously describedin) for the converting acetyl-CoA to malonyl-CoA and hence to malonatesemialdehyde and 3-HP. The present invention contemplates the use ofgenetic modifications that increase activity of ACCase complex enzymesto thereby increase 3-HP production in a host cell.

Fused Subunits

The acetyl-CoA carboxylase complex (ACCase) is a multi-subunit protein.Prokaryotes and plants have multi-subunit ACCs composed of severalpolypeptides encoded by distinct genes. However, humans and most othereukaryotes, such as yeast, have evolved an ACC with CT and BC catalyticdomains and biotin carboxyl carrier domains on a single polypeptide. Thebiotin carboxylase (BC) activity, biotin carboxyl carrier protein(BCCP), and carboxyl transferase (CT) activity are each contained on adifferent subunit. In E. coli the ACCase complex is derived from multipolypeptide transcribed by distinct, separable protein components knownas accA, accB, accC, and accD.

Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes theirreversible carboxylation of acetyl-CoA to produce malonyl-CoA throughits two catalytic activities, biotin carboxylase (BC) andcarboxyltransferase (CT). The first reaction is carried out by BC andinvolves the ATP-dependent carboxylation of biotin with bicarbonate. Thecarboxyl group is transferred from biotin to acetyl-CoA to formmalonyl-CoA in the second reaction, which is catalyzed by CT. The mainfunction of ACCase complex in the cell is to provide the malonyl-CoAsubstrate for the biosynthesis of fatty acids.

The conversion of acetyl-CoA to malonyl-CoA is an important step in thebioconversion of a renewable carbon source (such as, for example, sugaror natural gas) to a useful industrial chemical (such as, for example,3-hydroxypropionic acid (3-HP)). In certain organisms, such as E. colior yeast, the native ACCase expression from the chromosome alone isinsufficient to enable the organism to produce chemicals such as 3-HP ata rate to support a commercial scale operation. Overexpression of theACCase complex has been shown to provide some advantage [U.S. 12/891,760U.S. 12/891,790 U.S. 13/055,138].

Applicants have discovered that the introduction of an acetyl-CoAcarboxylase gene with one or more of its subunits fused is beneficial tothe production of a chemical product in a host cell. In certain aspectsof the invention, fusion is the two gene products produced from a singlepolypeptide controlled by a single promoter, will further enhance anorganism's bioproduction of an industrial chemical. In certain aspectsof the invention, fusion is the two gene products produced by at leastone promoter, will further enhance an organism's bioproduction of anindustrial chemical. In certain aspects of the invention, fusion is thetwo gene products produced from a single polypeptide controlled by atleast one inducible promoter, will further enhance an organism'sbioproduction of an industrial chemical. Keeping components of theACCase complex fused together in the genetic structure of an organismcan be advantageous because it enhances the stability of the non-nativeACCase genetic modification and it facilitates equimolar expression ofthe fused acc subunits.

In particular, the subunit-fused ACCase may be an accA-accB, accA-accC,accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC,accA-accB-accD, accA-accC-accD, accB-accC-accD or accA-accB-accC-accDfused subunit that have having at least 80% sequence homology to E. coliaccA, accB, accC and accD or is a functional homolog thereof. Inaddition, the organism may include any combination of these fusedsubunits, or any combination of these fused subunits together with oneor more of the four non-fused subunits. When such combinations are used,the subunits (fused and non-fused) may be expressed on the same plasmidor on different plasmids or on the chromosome of the organism.

In accordance with a preferred embodiment, an accA-accD fused subunit isintroduced into an organism either alone or in combination with theaccB-accC fused subunit, the accB gene, and/or the accC gene. Inaccordance with a preferred embodiment, the organism is a bacteria, andpreferably E. coli or Cupriavidus necator.

Composition Stoichiometry

Composition stoichiometry is the quantitative relationships amongelements that comprise a compound. A stoichiometric ratio of a reagentis the optimum amount or ratio where, assuming that the reactionproceeds to completion. Although stoichiometric terms are traditionallyreserved for chemical compounds, theses theoretical consideration ofstoichiometry are relevant when considering the optimal function ofheterologous multi-subunit protein in a host cell.

In accordance with another aspect of the invention, the stoichiometricratio of each of the four ACCase subunits relative to one another isimportant, and each such ratio can be between 0 and about 10, andpreferably between about 0.5 to about 2 or about 7 to about 9. Inaccordance with a preferred embodiment the ratios for the proteinsubunits are accA:accB:accC:accD are 1:2:1:1. In accordance with apreferred embodiment, an organism is genetically modified to include anaccA-accD fused subunit, an accB non-fused subunit, and an accCnon-fused subunit, with the molar ratios of the accDA fusion:accB:accCbeing about 1:2:1, which is close to the optimum for enzymatic activity.

In certain embodiments where an organism is engineered to make 3-HP, inorder to get optimal function in a host cell of a heterologous ACCaseenzyme complex it is important to engineer the stoichiometry of thesesubunits in such a way that provides maximal production of 3-HP suchthat the subunit can make a more stable enzyme complex whenoverexpressed in the cell.

In certain aspects the invention provides for the controlled expressionof the natural accA, accB, accC, and accD subunits of E. coli or havingat least 80% sequence homology to E. coli accA, accB, accC and accD. Incertain aspects the invention provides for the inducible expression ofthe natural accA, accB, accC, and accD subunits of E. coli or having atleast 80% sequence homology to E. coli accA, accB, accC, and accD. Incertain aspects the invention provides for the low, medium, high and/orinducible expression of the natural accA, accB, accC, and accD subunitsof E. coli or having at least 80% sequence homology to E. coli accA,accB, accC and accD.

In certain aspects the invention provides for the expression of thenatural accC and accD subunits of E. coli or having at least 80%sequence homology to E. coli accA, accB, accC and accD in low, medium,high or inducible expression. In certain aspects the invention providesfor the expression of the natural accB and accA subunits of E. coli orhaving at least 80% sequence homology to E. coli accA, accB, accC, andaccD in low, medium, high or inducible expression. In certain aspectsthe invention provides for the expression of the natural accC and accDsubunits with the accA subunit of E. coli or having at least 80%sequence homology to E. coli accA, accB, accC, and accD in low, medium,high or inducible expression. In certain aspects the invention providesfor the expression of the natural accC and accD subunits with the accBsubunit of E. coli or having at least 80% sequence homology to E. coliaccA, accB, accC, and accD in low, medium, high or inducible expression.

In certain aspects the invention provides for the expression of a fusionof two, three, or all of the four ACCase subunits in one polypeptide inlow, medium, high or inducible expression. Such fusion may include anyof the following combinations of the ACCase subunits: accA-accB,accA-accC, accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC,accA-accB-accD, accA-accC-accD, accB-accC-accD, and accA-accB-accC-accDhave having at least 80% sequence homology to E. coli accA, accB, accCand accD or is a functional homolog thereof.

In certain aspects the invention provides for ACC complex in thestoichiometry of these subunits of the accCB and accDA in a 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1,3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1,4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1,6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1,8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducibleexpression. In certain aspects the invention provides for ACC complex inthe stoichiometry of these subunits of the accDA and accCB in a 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8,3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8,4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8,6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8,8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducibleexpression.

In certain aspects the invention provides for the stoichiometry of theaccD-A subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3,2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3,3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3,5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3,7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low,medium, high or inducible expression. In certain aspects the inventionprovides for the stoichiometry of the accC-B subunits in a 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1,3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1,4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1,6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1,8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducibleexpression. In certain aspects the invention provides for thestoichiometry of the accC-A subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6,3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6,4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6,6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6,8:7, or 8:8 in low, medium, high or inducible expression. In certainaspects the invention provides for the stoichiometry of the accC-Bsubunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4,2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4,3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4,5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4,7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium,high or inducible expression.

-   -   III. CONVERSION OF MALONYL-COA TO MALONATE SEMIALDEHYDE

One of the steps in the biosynthesis of 3-HP involves the conversion ofmalonyl-CoA (MCA) to malonate semialdehyde (MSA) and the conversion ofmalonate semialdehyde (MSA) to 3-HP (WO2011/038364). In accordance withanother aspect of the present invention, the present inventioncontemplates the use of novel enzymes and/or combinations of enzymes tocatalyze the reaction in a microorganism from MCA to MSA, which resultsin enhanced cellular bioproduction of 3-HP in the host cell.

In certain aspects the invention provides novel enzyme compositions orco-expression of a combinations of enzyme compositions to catalyze theconversion of malonyl-CoA to 3-HP. A general overview of the enzymes andthe relevant reaction pathways methods are shown in FIG. 1.

In accordance with this aspect of the invention, malonyl-CoA isconverted to malonate semialdehyde by a malonyl-CoA reductase andmalonate semialdehyde is converted to 3-HP through either or both of twoalternative pathways.

In accordance with one aspect of the invention, malonyl-CoA is convertedto malonate semialdehyde by a monofunctional malonyl-CoA reductase thatcatalyzes the malonyl-CoA conversion, but does not catalyze the malonatesemialdehyde conversion.

In one embodiment, the microorganism herein comprise a geneticmodification that include the monofunctional malonyl-CoA reductase maybe derived from Sulfolobus tokodaii (stMCR) (SEQ ID NO. 15 nucleic acid,SEQ ID NO. 16 protein sequence) or a functional homolog of stMCR or ahomolog with at least 80% identity.

In some embodiments, the microorganism herein comprise a geneticmodification that include the bi-functional malonyl-CoA reductasecomprised of two protein fragments with one fragment having malonyl-CoAreductase activity and the other fragment having malonate semialdehydedehydrogenase activity may be derived from Chloroflexus aurantiacus(caMCR).

MCR-Dehydrogenase Enzymes for Conversion of 3-HP Ions

Following the conversion of the malonyl-CoA to malonate semialdehyde,the malonate semialdehyde is converted to 3-HP through either or both oftwo alternative pathways. Malonate semialdehyde may exist in at leastthree states; the keto form, the enol form, and hydrate form, as shownin FIG. 2. Malonate semialdehyde in the enol form, which will stabilizethis form when compared to other aldehydes where the enol form is highlyunfavored in the equilibrium among the three forms.

The malonate semialdehyde keto form is converted to 3-HP utilizing a3-hydroxy acid dehydrogenase enzyme (ydfG SEQ ID NO. 21 nucleic acid,SEQ ID NO. 22 protein), a 3-hydroxyisobutyrate dehydrogenase enzyme(Pseudomonas aeruginosa mmsB, SEQ ID No 23 nucleic acid, SEQ ID NO. 24protein), and/or NAD+-dependent serine dehydrogenase (Pseudomonas NDSD,SEQ ID NO. 25 nucleic acid, SEQ ID NO. 26 protein). In accordance with apreferred embodiment, Pseudomonas mmsB, Pseudomonas NDSD, and E. coliydfG are used. The gene, ydfG from E. coli is largely NADPH dependent,whereas mmsB and NDSD from Pseudomonas can utilize either NADPH or NADH.

The malonate semialdehyde enol form is converted to 3-HP utilizing anN-ethylmaleimide reductase (nemA, SEQ ID NO. 17 nucleic acid, SEQ ID NO.18 protein), and/or a malonic semialdehyde reductase (rutE, SEQ ID NO.19nucleic acid, SEQ ID NO. 20 protein) from E. coli. These enzymes doesnot directly utilize NADPH or NADH. Instead, these enzymes utilize aflavin mononucleotide that is cycled between oxidized and reduced statesby NADPH or NADH. The enol pathway also has advantages over the ketopathway in that equilibrium between the malonate semialdehyde enol formand 3-HP significantly favors 3-HP, making the reaction much lessreversible, and essentially irreversible.

The malonate semialdehyde hydrated form may also be converted to 3-HP byeither the 3-HP dehydrogenase or malonate semialdehyde reductaseenzymes, although the hydrated form is more likely to be converted tothe enol form as the equilibrium continuously readjusts.

In one embodiment, the microorganism herein comprise a geneticmodification that include (i.e., microorganism) includes apolynucleotide encoding: (1) a monofunctional malonyl-CoA reductase genecapable of catalyzing the conversion of malonyl-CoA to malonatesemialdehyde; and (2) one or more genes encoding one or more of thefollowing enzymes: ydfG, mmsB, NDSD, rutE, and nemA or a functionalhomolog or a homolog with at least 80% identity.

In accordance with another aspect of the invention, there is provided anorganism that is genetically modified to make 3-HP, wherein the geneticmodification includes a polynucleotide encoding: (1) a monofunctionalmalonyl-CoA reductase gene capable of catalyzing the conversion ofmalonyl-CoA to malonate semialdehyde; (2) one or more genes encoding oneor more enzymes capable of converting malonate semialdehyde keto form to3-HP; and (3) one or more genes encoding one or more enzymes capable ofconverting either the malonate semialdehyde enol form or the malonatesemialdehyde hydrated form to 3-HP.

In certain aspects the invention provides monofunctional malonyl-CoAreductase enzyme fused to a dehydrogenase enzyme that is either: (1)primarily not NADPH-dependent; (2) primarily NADH-dependent; (3)primarily flavin-dependent; (4) less susceptible to 3-HP inhibition athigh concentration; and/or (5) catalyzes a reaction pathway to 3-HP thatis substantially irreversible.

In certain aspects the invention also provides monofunctionalmalonyl-CoA reductase enzyme fused to a dehydrogenase enzyme that isNADPH-dependent.

Suitable 3-HP dehydrogenase enzymes that are largely NADH-dependent thatcan be used with the claimed invention include, but are not limited to,mmsB or NDSD. Suitable malonate reductase enzymes that areflavin-dependent include, but are not limited to, rutE and nemA.Suitable 3-HP dehydrogenase enzymes that are less susceptible 3-HPinhibition at high concentration that can be used with the claimedinvention include, but are not limited to, ydfG and NDSD. Suitable 3-HPdehydrogenase or malonate semialdehyde dehydrogenase enzymes thatcatalyze a reaction pathway to 3-HP that is substantially irreversibleare rutE and nemA.

In certain aspects the invention provides monofunctional malonyl-CoAreductase enzyme fused to one or more dehydrogenase enzymes. Malonatesemialdehyde, which is the intermediate product in the conversion ofmalonyl-CoA to 3-HP can be very reactive. Therefore, it is advantageousto have a reaction pathway wherein the residence time of malonatesemialdehyde within the cell is minimized, and its conversion to 3-HPoccurs quickly. By fusing the malonyl-CoA reductase with the malonatesemialdehyde dehydrogenase to create a multi-domain protein (e.g., twodomain protein) and having the MCR and dehydrogenase domains adjacent inthe sequence, when the themalonate semialdehyde is quicklyis quicklyconverted to 3-HP.

In certain aspects the invention provides first monofunctionalmalonyl-CoA reductase enzyme fused to a first dehydrogenase enzyme ofone type and second monofunctional malonyl-CoA reductase enzyme fused toa dehydrogenase enzyme of a different type than the first dehydrogenaseenzyme. Suitable different dehydrogenase enzymes include, but are notlimited to, enzymes that function on the different forms of malonatesemialdehyde.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that include but are not limited to themalonyl-CoA reductase from S. tokadaii is fused to ydfG, mmsB, NDSD,rutE, or nemA (or some combination thereof). The fused enzyme mayinclude any of the following configurations: mcr-ydfG, mcr-mmsB,mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD,mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD,mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB,mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB,mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB,mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with80% sequence identity thereof.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that include but are not limited to themalonyl-CoA reductase from C. aggregans is fused to ydfG, mmsB, NDSD,rutE, or nemA (or some combination thereof). The fused enzyme mayinclude any of the following configurations: mcr-ydfG, mcr-mmsB,mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD,mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD,mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB,mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB,mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB,mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with80% sequence identity thereof.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that include but are not limited to themalonyl-CoA reductase from O. trichoides is fused to ydfG, mmsB, NDSD,rutE, or nemA (or some combination thereof). The fused enzyme mayinclude but are not limited to any of the following configurations:mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB,mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG,mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG,mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG,mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG,mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog orhomolog with 80% sequence identity thereof.

Enhanced Mutated Monofunctional MCR for Bioproduction

In certain aspects the invention provides for microorganisms comprisinga genetic modification that include mutated form of stMCR that hasenhanced activity at about 20° C. to about 44° C., about 30° C. to about37° C., or about 32° C. to about 38° C. Such mutate forms may bedesigned based on the crystal structure now available for stMCR [Demmeret al., J. Biol. Chem. 288:6363-6370, 2013].

It is also contemplated the carboxylase domains of the malonyl-CoAreductase derived from Chloroflexus aggregans, Oscillochloris trichoidescan be enhanced by mutations in the carboxylase binding domain toprovide increased 3-HP production over the natural occurring enzyme.

The carboxylase activity of the malonyl-CoA reductase derived fromChloroflexus aurantiacus can be enhanced activity. In certain aspectsthe invention provides for mutated form of it carboxylase domain toprovide increased 3-HP production over the natural occurring enzyme.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that include carboxylase domains of themalonyl-CoA reductase derived from C. aggregans is fused to ydfG, mmsB,NDSD, rutE, or nemA (or some combination thereof). It is contemplatedthat the any of the enhanced MCR by mutation, as provide above, may befused in any of the following configurations including but not limitedto mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB,mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG,mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG,mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG,mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG,mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog orhomolog with 80% sequence identity thereof.

-   -   IV. SALT-TOLERANT ENZYMES

The growth of engineered microorganism for enhanced production of achemical product, such as E. coli is severely inhibited by high saltconcentrations accumulated when the chemical product is produced at highrate within the organism.

Dose-dependent studies with increasing amounts of NaCl and Na-3-HP showthat salt has inhibitory effects on ACCase activity which is essentialto fatty acid biosynthesis of membranes required for growth andpropagation and for the production of 3-HP (see EXAMPLE 1). Thus, theuse of salt-tolerant enzymes in 3-HP production should increase 3-HPproduction in a host cell.

A. Enzymes from Halophilic Organisms

Halophiles are characterized as organisms with a great affinity forsalt. In some instances a halophilic organism is one that requires atleast 0.05 M, 0.1 M, 0.2 M, 0.3 M or 0.4 M concentrations of salt (NaCl)for growth. Halophiles live in hypersaline environments that aregenerally defined occurring to their salt concentration of theirhabitats. Halophilic organisms that are defined as “Slight saltaffinity” have optimal growth at 2-5% NaCl, moderate halophiles haveoptimal growth at 5-20% NaCl and extreme halophiles have optimal growthat 20-30% NaCl.

Depending on the conditions of that the genetically engineeredmicroorganism is under one might use homologous enzymes of the inventionspecifically, for example, from a moderate halophiles or an extremehalophiles depending on the engineered cell's environment.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that includes enzymes of the invention providedherein from slight halophiles organisms. In certain aspects theinvention provides for microorganisms comprising a genetic modificationthat includes enzymes of the invention provided herein from moderatehalophiles organisms. In certain aspects the invention provides formicroorganisms comprising a genetic modification that includeshomologous enzymes of the invention provided herein from extremehalophiles organisms.

Homology with genes provided by the invention may be determined byanalysis with BLASTN version 2.0 provided through the NCBI website.Homology with proteins provided by the invention may be determined byanalysis with BLASTP version 2.2.2 provided through the NCBI website.This program with aligns the disclosed fragments being compared anddetermines the extent of identity or similarity between them.

To date there are many sequenced halophilic organisms which can be usedwith the claimed invention. Examples of some sequenced halophilicorganisms include but are not limited to Chromohalobacter salexigens,Flexistipes sinusarabici strain (MAS10T), Halobacterium sp. NRC-1,Haloarcula marismortui, Natronomonas pharaonis, Haloquadratum walsbyi,Haloferax volcanii, Halorubrum lacusprofundi, Halobacterium sp. R-1,Halomicrobium mukohataei, Halorhabdus utahensis, Halogeometricumborinquense, Haloterrigena turkmenica, Natronobacterium gregoryi SP2,Halalkalicoccus jeotgali, Natrialba magadii, Haloarcula hispanica,Halopiger xanaduensis, Halophilic archaeon DL31, Haloferax mediterranei,Halovivax ruber, Natronococcus gregoryi, and Natronococcus occultus.

Examples of suitable moderate halophilic organisms in which homologousenzymes of the invention can be derived from include but are not limitedto eukaryotes such as crustaceans (e.g. Artemia salina), insects (e.g.Ephydra hians), certain plants from the genera Salicornia spp, algae(e.g. Dunaliella viridis), fungi, and protozoa (e.g. Fabrea salina),phototrophic organisms, such as planktonic and microbial mat-formerscyanobacteria as well as other anaerobic red and green sulphur bacteriafrom the genera Ectothiorhodospira spp.) and non-sulphur bacteria fromthe genera Chromatium spp.; gram-negative anaerobic bacteria, forexample from the genera Haloanaerobacter spp. some of which aremethanogenic, for example from the genera Methanohalophilus spp. andeither aerobic or facultative such as species from the genera Halomonas,Chromohalobacter, Salinovibrio, Pseudomonas, for example (e.g.Halomonase elongate); gram-positive bacteria from genera such asHalobacillus, Bacillus, Marinococcus, etc. as well as someactinomycetes, for example, Actinopolyspora halophila.

Genomic and Proteomic Hallmarks of Halophilic Organisms

Comparative genomic and proteomic studies of halophiles andnon-halophiles reveal some common trends in the genomes and proteomes ofhalophiles. At the protein level, halophilic organisms are characterizedby low hydrophobicity, over-representation of acidic residues,especially Asp, under-representation of Cys, lower propensities forhelix formation and higher propensities for coil structure.

At the DNA level, halophilic organisms are characterized by thedinucleotide abundance profiles of halophilic genomes bear some commoncharacteristics, which are quite distinct from those of non-halophiles,and hence may be regarded as specific genomic signatures forsalt-adaptation. The synonymous codon usage in halophiles also exhibitssimilar patterns regardless of their long-term evolutionary history.

In certain aspects the invention provides for microorganisms comprisinga genetic modification that the proteins provided by the invention thatare modified for salt tolerance such that they has low hydrophobicity,over-representation of acidic residues, especially Asp,under-representation of Cys, lower propensities for helix formation andhigher propensities for coil structure.

Suitable salt-tolerant enzymes can include enzymes from salt-tolerantorganisms. Salt-tolerant organisms (such as, for example, halophiles)include any living organism that are adapted to living in conditions ofhigh salinity. Suitable salt-tolerant enzymes can include enzymes fromsalt-tolerant organism that are homologs of the following enzymes:Sucrose-6-phosphate hydrolase (cscA from E. coli), glucose-6-phosphateisomerase (pgi from E. coli), fructokinase (cscK from E. coli),fructose-1,6-bisphosphatase (yggF from E. coli), fructose1,6-bisphosphatase (ybhA from E. coli), fructose 1,6-bisphosphatase II(glpX from E. coli), fructose-1,6-bisphosphatase monomer (fbp from E.coli), 6-phosphofructokinase-1 monomer (pfkA from E. coli),6-phosphofructokinase-2 monomer (pfkB from E. coli), fructosebisphosphate aldolase monomer (fbaB from E. coli), fructose bisphosphatealdolase monomer (fbaA from E. coli), triose phosphate isomerase monomer(tpiA), glyceraldehyde 3-phosphate dehydrogenase-A monomer (gapA from E.coli), phosphoglycerate kinase (pgk),2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmM fromE. coli), 2,3-bisphosphoglycerate-dependent or tdcE (from E. coli),phosphoglycerate mutase (gpmA), enolase (eno from E. coli),phosphoenolpyruvate carboxylase (ppc from E. coli), malate dehydrogenase(mdh), fumarase A (fum from E. coli), fumarase B (fumB), fumarase C(fumC from E. coli), phosphoenolpyruvate synthetase (ppsA from E. coli),pyruvate kinase I monomer (pykF from E. coli), pyruvate kinase IImonomer (pykA from E. coli), fumarate reductase (frdABCD from E. coli),lipoamide dehydrogenase (lpd from E. coli), pyruvate dehydrogenase (aceEfrom E. coli), pyruvate dehydrogenase (aceF from E. coli), pyruvateformate-lyase (pflB from E. coli), acetyl-CoA carboxylase (accABCD fromE. coli), malonyl CoA reductase (mcr), 3HP dehydrogenase (mmsB, NDSD, orydfG), malonate semialdehyde reductase (nemA, rutE from E. coli), or acombination thereof.

Suitable salt-tolerant enzyme homologs that can be used with the claimedinvention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%, overallamino acid or nucleotide identity to the above enzymes. Suitablesalt-tolerant enzyme homologs that can be used with the claimedinvention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%,90% 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%, amino acidor nucleotide to the essential protein function domains of the enzymesabove. Suitable salt-tolerant enzyme homologs that can be used with theclaimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overallamino acid or nucleotide to the essential binding amino acids within anessential protein function domain of the enzymes above.

In accordance with a preferred embodiment of the invention, suitablesalt-tolerant enzyme homologs are enzymes from one of the followingorganisms: Halomonas elongata, Salinibacter rubur, or Halobacteriumspecies (Archaea).

In accordance with a preferred embodiment of the present invention,there is provided a non-salt-tolerant organism that is geneticallymodified to make 3-HP, wherein the genetic modification includes apolynucleotide encoding an acetyl-CoA carboxylase from a salt-tolerantorganism. In accordance with a preferred embodiment, the acetyl-CoAcarboxylase subunits accA, accB, accC and accD is from Halomonaselongata.

-   -   V. CHEMICAL TRANSPORTER

In accordance with another aspect of the present invention, any of themicroorganisms herein may be genetically modified to introduce a nucleicacid sequence coding for a polypeptide that: (1) facilitates theexportation of the chemical of interest or the export of an inhibitorychemical from within the cell to the extracellular media; and/or (2)facilitates the importation from the extracellular media to within thecell of a reactant, precursor, and/or metabolite used in the organism'sproduction pathway for producing the chemical of interest.

3-HP Exporter

In accordance with a preferred embodiment, this invention relates to thebioproduction of 3-HP using a genetically modified E. coli organism.Thus, the present invention contemplates of a host cell geneticallymodified to express or increase expression of an exporter that canfunction to transfer 3HP from the cellular environment extracellularly.

Bacterial cells, such as E. coli, have at least five different types ofexporters: the major facilitator superfamily (MFS); the ATP-bindingcassette superfamily (ABC); the small multidrug resistance family (SMR);the resistance-nodulation-cell division superfamily (RND); and the multiantimicrobial extrusion protein family (MATE). In addition, amino acidexporters, which are common to almost all host cells, are likely toexport 3-HP. Additionally, solvent tolerance transporters, for examplebromoacetate, butanol, isobutanol and the alike may be used to export3-HP.

In certain aspects the invention provides a host cell with a recombinantexporter wherein the exporter is an MFS exporter, ABC exporter, SMRexporter, RND exporter, MATE exporter, amino acid exporter, solventtolerance transporter or a combination thereof.

Suitable exporters that can be used with the s herein invention includebut are not limited to acrD, bcr, cusA, dedA, eamA, eamB, eamH, emaA,emaB, emrB, emrD, emrKY, emrY, garP, gudP, hsrA, leuE, mdlB, mdtD, mdtG,mdtL, mdtM, mhpT, rhtA, rhtB, rhtC, thtB, yahN, yajR, ybbP, ybiF, ybjJ,ycaP, ydcO, yddG, ydeD, ydgE, yddG, ydhC, ydhP, ydiN, ydiM, ydjE, ydjl,ydjK, yeaS, yedA, yeeO, yegH, yggA, yfcJ, yfiK, yhjE, yidE, yigK, yigJ,yijE, yjiI, yjiJ, yjiO, ykgH, ypjD, ytfF, ytfL or functional homolog orhomolog with 80% sequence identity thereof. Other potential transporterproteins may be identified using topology analysis as illustrated in[Daley et al., Science 308: 1321-1323, 2005].

In certain aspects the invention provides the exporter to be improvedfor binding to 3-HP. In certain aspects the invention provides theexporters named to be further enhance by genetic modification of thepredicted cytoplasmic domain to increase 3-HP binding. In certainaspects the invention provides the exporter to be improved for bindingto 3-HP. In certain aspects the invention provides the exporters namedto be further enhance by genetic modification of the predictedtransmembrane binding domain to increase 3-HP binding or incorporationinto the host cell membrane.

Suitable exporter homologs that can be used with the claimed inventioncan have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%,88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid ornucleotide identity to the above exporters. Suitable exporter homologsthat can be used with the claimed invention can have 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%,90%,89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%or 80% amino acid or nucleotide to the essential protein functiondomains of the exporters above. Suitable exporter homologs that can beused with the claimed invention can have at least 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,81% or 80% overall amino acid or nucleotide to the essential bindingamino acids within an essential exporter domain of the enzymes above.

In certain aspects the invention provides for at least of the exportersprovided herein to be expressed in a host cell to increase the chemicalproduction of 3-HP in a host cell. In certain aspects the inventionprovides for at least of the exporters provided herein to be expressedin a host cell and with a genetic modification of tig to increase thechemical production of 3-HP in a host cell.

In certain aspects the invention provides for one exporter to be furthermodified by on one more genetic modulates so that the expression leveland timing of expression of the exporter can be controlled in the hostcell. In certain aspects the invention provides for one exporter to befurther modified by an inducible promoter, RBS, high, multicopy plasmidor combination thereof, as provide herein, in order to control itsexpression in the host cell.

In certain aspects the invention provides exporters provide herein to beexpressed in a host cell in equal ratio. In certain aspects theinvention provides exporters provide herein to be expressed in a hostcell in equal 1:2 ratio. In certain aspects the invention providesexporters provide herein to be expressed in a host cell in equal 1:3ratio. In certain aspects the invention provides exporters provideherein to be expressed in a host cell in equal 1:4 ratio. In certainaspects the invention provides exporters provide herein to be expressedin a host cell in equal 2:3 ratio.

In certain aspects the invention provides for the exporter to maintainthe host cell at pH 7.0-7.4 during the continuous production phase. Incertain aspects the invention provides for the exporter and the meansfor importing a base inside the cell in order to maintain the host cellat pH 7.0-7.4 during the continuous production phase. In certain aspectsthe invention provides for the exporter maintain the host cell at pH 3.0to pH 4.0, pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0to pH 8.0, pH 8.0 to pH 9.0, or pH 9.0 to pH 10.0 pH 7.0-7.3 during thecontinuous production phase. In certain aspects the invention providesfor the exporter and the means for importing a base inside the cell inorder to maintain the host cell at pH 3.0 to pH 4.0, pH 4.0 to pH 5.0,pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 to pH 9.0,or pH 9.0 to pH 10.0 pH 7.0-7.3 during the continuous production phase.

In accordance with this aspect of the present invention, additionmodifications to the host cell may be made to further enhance thetransporter's function. In particular, deletion of the tig gene from thegenome of the host cell may enhance expression and total activity ofintegral membrane proteins such as exporters and importers.

Bicarbonate Importer

One of the key steps in the conversion of biomass to 3-HP is theconversion of acetyl-CoA to malonyl-CoA, which is illustrated in FIG. 3.

As shown in FIG. 3, this reaction is catalyzed by the acetyl-CoAcarboxylase, and bicarbonate is a reactant needed to drive the reaction.One of the primary sources of bicarbonate to drive this reaction iscarbon dioxide within the cell. Carbon dioxide is readily diffusiblethrough a cell's membrane, and a natural equilibrium will be reachedbetween the intracellular and extracellular carbon dioxide. As a cellproduces carbon dioxide it migrates through the cell, and since it isnot very soluble in the media, it will bubble out of the system and moreintracellular carbon dioxide will migrate out of the cell to maintainthe equilibrium. This process impedes the production of 3-HP sincebicarbonate (which is in equilibrium with the dissolved carbon dioxidein the form of carbonic acid) is needed to drive theacetyl-CoA→malonyl-CoA reaction, and the intracellular carbon dioxide isthe primary source for intracellular bicarbonate.

In accordance with one aspect of the present invention, an organism isprovided that includes a heterologous gene encoded therein that acts asa carbon dioxide importer (i.e., it enhances the importation of carbondioxide into the cell or inhibits the exportation of carbon dioxide fromthe cell), which results in increased intracellular carbon dioxide. Useof CO2an importer mitigates against the natural outflow of carbondioxide.

In accordance with this aspect of the invention, there is provided anorganism that is genetically modified, wherein the genetic modificationincludes a polynucleotide encoding a gene capable of importingextracellular carbon dioxide from the media to within the cell membraneor inhibiting the exportation of intracellular carbon dioxide fromwithin the cell membrane to the media. In accordance with a preferredembodiment of the present invention, a microorganism is geneticallymodified to encode one or more of the following heterologous genes: bicAfrom Synechococcus species, ychM gene product of E. coli, yidE geneproduct of E. coli, any of the bicarbonate transporters as described in[Felce and Saier, J. Mol. Microbiol. Biotechnol. 8: 169-176, 2004 or anyamino acid sequences homologous thereof (e.g., at least 80%, 85%, 90%,95%, or 99% homologous to the amino acids sequences of the CO2importer/exporters described herein].

Bioproduction Methods

In some applications of the invention the host cell is geneticallymodified for increased malonyl-CoA flux by at least one heterologousACCase complex, such as Table 4 to further increase chemicalbio-production in host cell. In some applications of the invention thehost cell is genetically modified with heterologous salt tolerantenzymes, such as Table 5 to increase chemical bio-production in a hostcell. In some applications of the invention the host cell is geneticallymodified with heterologous 3-HP exporters to further increase chemicalbio-production in a host cell.

In some applications of the invention the host cell is geneticallymodified by at least one heterologous gene and/or salt tolerantheterologous gene of FIG. 1 or Table 5 and at least one 3-HP exporterprovided herein to further increase chemical bioproduction in a hostcell.

In some applications of the invention the host cell is geneticallymodified with a heterologous gene for increased malonyl-CoA flux, 3-HPexport, at least one heterologous and/or salt tolerant heterologousgene, as provided herein, to increase chemical bio-production in a hostcell. In some applications of the invention the host cell is geneticallymodified for increased malonyl-CoA flux, 3-HP export, at least oneheterologous gene and/or salt tolerant heterologous gene and the hostcell is genetically modified by at least one gene, as provided herein toincrease chemical bioproduction in a host cell.

When utilizing certain organisms to create certain products, it may beadvantageous to control each phase discretely. For example, depending onthe pathway involved, reactions, reactants, intermediates and byproductscreated during cell growth can inhibit enzyme induction and/or theorganism's ability to produce the desired chemical product. Similarly,reactions, reactants, intermediates and byproducts created as part ofthe production pathway can impact cell growth, and even the increasedconcentration of the chemical product as it is produced can impede cellreplication.

Table. 5

-   -   VI. MULTI-PHASE FERMENTATION

In accordance with another aspect of the present invention, there isprovided a method of producing a chemical product from a carbon sourcethrough a bioproduction process that comprises a controlled multi-phaseproduction process. The multi-phase production process includes aninitiation and/or completion of one or more phases of the productionprocess is controlled by genetic modifications to the organism producingthe chemical product and/or is controlled by changes made to the cellenvironment.

In accordance with this aspect of the invention, the bioproductionprocess may include two or more of the following phases: (1) growthphase; (2) induction phase; and (3) production phase. During the growthphase, the organism replicates itself and the biocatalyst needed toproduce the chemical product is built up. During the induction phase,expression of key enzymes critical to the production of the chemical isinduced and the enzymes accumulate within the biocatalyst to carry outthe reactions required to produce the product. During the productionphase organism produces the desired chemical product.

The initiation and/or completion of the growth, induction and/orproduction phases are controlled. In accordance with the presentinvention, the growth phase is dependent on the presence of a criticalexternal reactant that will initiate growth. The initiation andcompletion of the growth phase is controlled by the addition and amountof the initiating reactant added to the reaction vessel.

In accordance with certain preferred embodiments of the presentinvention, the chemical product is 3-HP and the production organism isE. coli or yeast. The critical external reactant may be phosphate, whichis needed for replication of E. coli cells. In accordance with apreferred embodiment, the growth phase is initiated by the addition ofphosphate to a reaction vessel (together with a carbon source such assugar and the E. coli cells), and the duration of the growth phase iscontrolled by the amount of phosphate added to the system.

The induction phase is controlled by genetic modifications to theproducing organism. The key enzymes triggered during this phase areengineered into the organism using promoters that are sensitive to(e.g., activated by) the depletion of the initiating reactant. As aresult, once the initiating reactant is depleted, the growth phase ends,the key enzymes are activated and the induction phase begins.

In accordance with a preferred embodiment, the induction phase iscontrolled by key genes that encode for enzymes in the biosyntheticpathway for the product into the production organism using promotersthat are activated by phosphate depletion. In one embodiment where thechemical product is 3-HP and the production organism is E. coli, the keygenetic modifications may include one or more of the following: mcr,mmsB, ydfG, rutE, nemA and NDSD; genes that encode individual or fusedsubunits of ACCase, such as accA, accB, accC, accD, accDA fusion, andaccCB fusion, and the promoters may include one or more of the promotersthat direct expression of the following E. coli genes: amn, tktB, xasA,yibD, ytfK, pstS, phoH, phnC, or other phosphate-regulated genes asdescribed in [Baek and Lee, FEMS Microbiol Lett 264: 104-109, 2006]. Inaccordance with this embodiment, once the phosphate is depleted,expression of the key enzymes is activated by their promoters and theinduction phase begins.

The production phase may also be controlled by genetic modifications.For example, the organism can be engineered to included mutated forms ofenzymes critical to the initiation of production of the chemicalproduct. These initiation enzymes may facilitate initiation ofproduction either by: (1) becoming active and serving a key function inthe production pathway; and/or (2) becoming inactive and thereby turningoff a branch pathway or other competitive pathway that prevents orlimits the production pathway leading to the chemical product. Inaccordance with a preferred embodiment, initiation enzymes are mutatedto form temperature sensitive variants of the enzymes that are eitheractivated by or deactivated at certain temperatures. As a result, theproduction phase is initiated by changing the changing the temperaturewithin the reaction vessel.

In one embodiment, the production phase is controlled by geneticallymodifying the microorganism with a heterologous nucleotide sequenceencoding i one or more of the following temperature sensitive enzymes:fabI^(ts) (SEQ ID NO. 27), fabB^(ts) (SEQ ID NO.28) and fabD^(ts) (SEQID NO. 29). These enzymes are deactivated or shut-off at the desiredtemperature for production of the chemical product. These enzymes play akey role shuttling carbon atoms into the fatty acid synthesis pathway.Although fatty acid synthesis pathway is critical during the growthphase, it inhibits production of the chemical product. Once the reactionvessel temperature is changed, the temperature sensitive enzymes aredeactivated and the fatty acid synthesis pathway shuts down therebyallowing the production pathway of the chemical product to ramp up.

In accordance with the present invention, the growth phase can last bebetween 10 to 40 hours, or about 15 to about 35 hours, or about 20 toabout 30 hours. The induction phase may be for about 1 to about 6 hours,about 1 to about 5 hours, or about 2 to about 4 hours. The productionphase may be greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100 hours depending on the amount ofchemical product that is desired.

In accordance with the present invention, the growth phase and inductionphase are conducted at a temperature of about 25° C. to about 35° C.,about 28° C. to about 32° C., or about 30° C. The production phase isconducted at a temperature of about 35° C. to about 45° C., about 35° C.to about 40° C., or about 36° C. to about 38° C. Preferably, theproduction phase temperature is higher than the induction phasetemperature, and the increase in temperature that initiates theproduction phase occurs over a period of about 1 to about 5 hours, about1 to about 3 hours, about 2 hours, or about 1 hour.

In accordance with the present invention, there is provided a method ofproducing a chemical product from a renewable carbon source through abioproduction process comprising:

-   -   (1) constructing a genetically modified organism capable of        converting said renewable carbon source to said chemical        product, wherein said genetically modified organism requires        inorganic phosphate for growth and comprises: (a) at least one        heterologous gene whose expression is regulated by a promoter        sensitive to inorganic phosphate levels within a culture system,        wherein said gene provides a critical function in converting        said carbon source to said chemical product and is not required        for the genetically modified organism to replicate; and (b) a        gene encoding a temperature- sensitive enzyme;    -   (2) forming a culture system comprising said carbon source in an        aqueous medium and said genetically modified microorganism;    -   (3) maintaining the culture system under conditions that allow        the genetically modified microorganism to replicate comprising        maintaining a sufficient level of inorganic phosphate within        said culture system;    -   (4) allowing the inorganic phosphate to deplete thereby        triggering the expression of the gene regulated by a promoter        sensitive to inorganic phosphate levels; and    -   (5) changing the temperature of the culture system thereby        activating or deactivating said temperature-sensitive enzyme and        initiating the production of said chemical product.

In accordance with the present invention, there is provided a method ofproducing 3-hydropropionic acid (3-HP) from a renewable carbon source,comprising:

-   -   (1) constructing a genetically modified organism capable of        converting said renewable carbon source to 3-HP, wherein said        genetically modified organism requires inorganic phosphate for        growth and comprises: (a) at least one heterologous gene whose        expression is regulated by a promoter sensitive to inorganic        phosphate levels within a culture system, wherein said gene is        selected from the group consisting of mcr, mmsB, ydfG, rutE,        nemA, NDSD, accA, accB, accC, accD, accDA fusion, and accCB        fusion; and (b) a gene encoding a temperature-sensitive enzyme        selected from the group consisting of fabI, fabB and fabD;    -   (2) forming a culture system comprising said carbon source in an        aqueous medium, phosphate and said genetically modified        microorganism, and thereby initiating a growth phase during        which the genetically modified microorganism replicates;    -   (3) maintaining a sufficient level of inorganic phosphate within        said culture system until the desired level of cell growth is        achieved;    -   (4) allowing the inorganic phosphate to deplete thereby        initiating an induction phase which begins the expression of        said gene regulated by a promoter sensitive to inorganic        phosphate levels; and    -   (5) changing the temperature of the culture system thereby        activating or deactivating said temperature-sensitive enzyme and        initiating a growth phase during which said genetically modified        microorganism produces 3-HP.

Fermentation Conditions

Depending on the host cell fermentation may be performed under aerobic,microaerobic, or anaerobic conditions, with or without agitation. Theoperation of culture systems to achieve aerobic, microaerobic andanaerobic conditions are well known to those of ordinary skill in theart.

Suitable pH ranges for fermentation depend on the multiple factors suchas the host cell. In some applications of the invention fermentation canoccur between various pH ranges for example, pH 3.0 to pH 4.0, pH 4.0 topH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 topH 9.0, or pH 9.0 to pH 10.0. However, the actual pH conditions for aparticular application are not meant to be limited by these ranges andcan be between the expressed pH ranges if it provides more optimalproduction of the fermentation process, such as increased 3-HPproduction.

-   -   VII. GENES AND PROTEINS FOR THE BIOPRODUCTION OF CHEMICALS

An overview of the engineered pathways provided by the invention in ahost cell is shown in FIG. 1. Various combinations of the pathways showncan be carried out by various combinations of genetic modifications tokey enzymes either in the intrinsic pathways or supplied through thetransformation of a heterologous gene.

In some applications of the genetically modified microorganism of theinvention may comprise a single genetic modification, or one or moregenetic modifications. Various types of genetic modifications that canbe used with the invention are disclosed herein.

In some embodiments the genetic modified organism of the invention cancomprise a genetic modification to the following gene/proteins or ahomolog with at least 80% identity to or a functional homolog of:bifunctional malonyl-CoA reductase (MCR from Chloroflexus aurantiacus),monofunctional malonyl-CoA reductase (caMCR from Chloroflexusaurantiacus), malonyl-CoA reductase (stMCR from Sulfolobus tokodaii.),Enzyme: malonyl-CoA reductase (cgMCR from Chloroflexus aggregans),Enzyme: malonyl-CoA reductase (otMCR from Oscillochloris trichoides),Polypeptide: host restriction; endonuclease R (hsdR from E. coli),lactose metabolism (lac from E. coli), L-rhamnulose kinase (rhaB from E.coli), rhamnulose-1-phosphate aldolase (rhaD from E. coli), Enzyme:β-galactosidase (lacZ from E. coli), L-ribulose 5-phosphate 4-epimerase(araD from E. coli), L-ribulokinase (araB from E. coli), Enzyme:D-lactate dehydrogenase-fermentative (ldhA from E. coli), enzyme:pyruvate formate-lyase (pflB from E. coli), Enzyme: phosphateacetyltransferase/phosphate propionyltransferase (pta from E. coli),Enzyme: pyruvate oxidase (poxB from E. coli), Enzyme: methylglyoxalsynthase (mgsA from E. coli), enzyme: Acetate kinase (ackA from E.coli), enzymes: phosphotransacetylase-acetate kinase (pta-ack from E.coli), Enzyme: enoyl-[acyl-carrier-protein] reductase (fabI from E.coli), Protein: zeocin binding protein (zeoR from StreptoalloteichusHindustanus), Enzyme: carboxytransferase moiety of acetyl-CoAcarboxylase (accAD from E. coli), Enzyme: triose phosphate isomerase(tpiA from E. coli), Enzyme: biotoin carboxylase moiety of acetyl-CoAcarboxylase (accBC from E. coli), Enzyme: transhydrogenase (pntAB fromE. coli), Polypeptide: LacI DNA-binding transcriptional repressor (lacIfrom E. coli), Enzyme: β-ketoacyl-ACP synthases I (fabB from E. coli),Enzyme: β-ketoacyl-ACP synthases II (fabF from E. coli), Enzyme:malonyl-CoA-ACP transacylase (fabD from E. coli), Enzyme: pantothenatekinase (coaA from E. coli), Enzyme: pyruvate dehydrogenase complex(aceEF from E. coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase(mmsB from Pseudomonas aeruginosa), Enzyme: lipoamide dehydrogenase (lpdfrom E. coli), Enzyme: γ-glutamyl-γ-aminobutyraldehyde dehydrogenase(puuC from E. coli), Enzyme: malate synthase A (aceB from E. coli),Enzyme: isocitrate lyase (aceA from E. coli), Enzyme: isocitratedehydrogenase phosphatase/kinase (aceK from E. coli), Enzyme: 3-hydroxyacid dehydrogenase (ydfG from E. coli), Enzyme: acetyl CoA carboxylase(accADBC from E. coli), Polypeptide: predicted transcriptional regulator(yieP from E. coli), Blastocyin resistance gene (BSD fromSchizosaccharomyces pombe), Enzyme: pyridine nucleotide transhydrogenase(udha from E. coli), Protein: Cra DNA-binding transcriptional dualregulator (fruR from E. coli), (SCB from E. coli), enzyme: aldehydedehydrogenase B (aldB from E. coli), Enzyme: carbonic anhydrase (cynTfrom E. coli), Enzyme: cyanase (cynS from E. coli), DNA gyrasetoxin-antitoxin system (ccdAB from E. coli), Enzyme: phosphoglyceratemutase (pgi from E. coli), ArcA transcriptional dual regulator orAerobic respiration control (arcA from E. coli), Enzyme:6-phosphofructokinase (pfk from E. coli), Enzyme: glyceraldehyde3-phosphate dehydrogenase-A complex (gapA from E. coli), aldehydedehydrogenase A (alda from E. coli), Enzyme: glutamate dehydrogenase(gdhA from E. coli), Enzyme: NADH-dependent serine dehydrogenase (NDSDfrom Pseudomonas aeruginosa), Protein: threonine/homoserine effluxtransporter (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphatedehydrogenase (gapN from E. coli), Phosphotransferase system (pts fromE. coli), Enzyme: 6-phosphofructokinase II (pfkB from E. coli), Enzyme:methylmalonate-semialdehyde dehydrogenase (mmsA from Pseudomonasaeruginosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 fromBacillus cereus), Enzyme: aspartate 1-decarboxylase (panD from E. coli),Gene that confers resistance to valine (ValR from E. coli), Enzyme:glucokinase (glk from E. coli), Polypeptide: 30 S ribosomal sununitprotein S12 (rpsL from E. coli), Polypeptide: CynR DNA-bindingtranscriptional repressor (cynR from E. coli), Transporter: galactose:H+symporter (galP from E. coli), aspartate aminotransferase (aspC from E.coli), Enzyme: alpha-ketoglutarate reductase (serA from E. coli),Enzyme: 6-phosphofructokinase I (pfkA from E. coli), Enzyme:phosphoenolpyruvate carboxylase (ppc from E. coli), Enzyme:succinate-semialdehyde dehydrogenase (NADP+) (gabD from E. coli),Enzyme: pyruvate kinase (pyk from E. coli), Enzyme: oxaloacetate4-decarboxylase (OAD from Leuconostoc mesenteroides), Enzyme: triggerfactor; a molecular chaperone involved in cell division (tig from E.coli), Transcription Unit (ptsHIcrr from E. coli), Enzyme: acetyl-CoAacetaldehyde dehydrogenase/alcohol dehydrogenase (adhE from E. coli),Enzyme: fattyacyl thioesterase I (tesA from E. coli), Enzyme: guanosine3′-diphosphate 5′-triphosphate 3′-diphosphatase (spoT from E. coli),combination of genes encoding accABCD subunits (from E. coli andHalomonas elongata), pol (from E. coli), Enzyme: GDPpyrophosphokinase/GTP pyrophosphokinase (relA from E. coli), [EnzymeName] (me from E. coli), Enzyme: citrate synthase (gltA from E. coli),Polypeptide: DNA gyrase, subunit A (gyrA from E. coli), Enzyme:multifunctional 2-keto-3-deoxygluconate 6-phosphate aldolase and2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (edafrom E. coli), thiamin biosynthesis (thi from E. coli), Polypeptide:acetolactate synthase II (ilvG from E. coli), acetyl CoA carboxylase(accDACB from E. coli), Citrate synthase (ArCS from Arthrobacteraurescens), Acetyl-CoA carboxylase from Corynebacter glutamicum (CgACCfrom Corynebacter glutamicum), Polypeptide: ferrichrome/phage/antibioticouter membrane porin FhuA (fhuA from E. coli), Transporter: phosphate:H+symporter PitA (pitA from E. coli), Transporter: uracil:H+ symporter(uraA from E. coli), Enzyme: uracil phosphoribosyltransferase (upp fromE. coli), Enzyme: acylphosphatase (yccX from E. coli), acetyl-CoAsynthetase (acsA from E. coli), Polypeptide: restriction of methylatedadenine (mrr from E. coli), Protein: TrpR transcriptional repressor(trpR from E. coli), Enzymes: glutamate 5-semialdehydedehydrogenase/gamma-glutamyl kinase (proAB from E. coli), methylcytosinerestriction system (mcrBC from E. coli), Protein: citrate lyase,citrate-ACP transferase component (citF from E. coli), Enzyme:thioesterase II (tesB from E. coli), Enzyme: DNA-specific endonuclease I(endA from E. coli), Enzyme: phosphate acetyltransferase (eutD from E.coli), Enzyme: propionate kinase (tdcD from E. coli), tRNA: tRNA glnV(supE from E. coli), Enzyme: DNA-binding, ATP-dependent protease La (lonfrom E. coli), Polypeptide: DNA strand exchange and recombinationprotein with protease and nuclease activity (recA from E. coli),Transcription Unit: restriction endonulease component of EcoKIrestriction-modification system (hsdRMS from E. coli), Enzyme:restriction of DNA at 5-methylcytosine residues (mcrA from E. coli) araD(from E. coli), araB (from E. coli), rhaD (from E. coli), rhaB (from E.coli), ack (from E. coli), fruR (from E. coli), gapA (from E. coli), lad(from E. coli), lacZ (from E. coli), ldhA (from E. coli), mgsA (from E.coli), pfkA (from E. coli), pflB (from E. coli), pgi (from E. coli),poxB (from E. coli), pta-ack (from E. coli), ptsH (from E. coli), glutl(from E. coli) and/or ack (from E. coli) or any combination thereof.

The use of genetic modifications in genetic elements, genes, proteins orthe use of compounds, such as siRNA technology, anti-sense technology,and small molecule inhibitors supplied to the host cell that modulatethe expression of gene and proteins provided by the present inventionare also contemplated.

In some embodiments the genetic modified organism of the invention usesgenetic elements such as siRNA ect, genes, proteins or compoundssupplied to the host cell to modulate one or more of the following:bifunctional malonyl-CoA reductase (MCR from Chloroflexus aurantiacus),monofunctional malonyl-CoA reductase (caMCR from Chloroflexusaurantiacus),malonyl-CoA reductase (stMCR from Sulfolobus tokodaii.),Enzyme: malonyl-CoA reductase (cgMCR from Chloroflexus aggregans),Enzyme: malonyl-CoA reductase (otMCR from Oscillochloris trichoides),Polypeptide: host restriction; endonuclease R (hsdR from E. coli),lactose metabolism (lac from E. coli), L-rhamnulose kinase (rhaB from E.coli), rhamnulose-1-phosphate aldolase (rhaD from E. coli), Enzyme:β-galactosidase (lacZ from E. coli), L-ribulose 5-phosphate 4-epimerase(araD from E. coli), L-ribulokinase (araB from E. coli), Enzyme:D-lactate dehydrogenase-fermentative (ldhA from E. coli), enzyme:pyruvate formate-lyase (pflB from E. coli), Enzyme: phosphateacetyltransferase/phosphate propionyltransferase (pta from E. coli),Enzyme: pyruvate oxidase (poxB from E. coli), Enzyme: methylglyoxalsynthase (mgsA from E. coli), enzyme: Acetate kinase (ackA from E.coli), enzymes: phosphotransacetylase-acetate kinase (pta-ack from E.coli), Enzyme: enoyl-[acyl-carrier-protein] reductase (fabI from E.coli), Protein: zeocin binding protein (zeoR from StreptoalloteichusHindustanus), Enzyme: carboxytransferase moiety of acetyl-CoAcarboxylase (accAD from E. coli), Enzyme: triose phosphate isomerase(tpiA from E. coli), Enzyme: biotoin carboxylase moiety of acetyl-CoAcarboxylase (accBC from E. coli), Enzyme: transhydrogenase (pntAB fromE. coli), Polypeptide: LacI DNA-binding transcriptional repressor (lacIfrom E. coli), Enzyme: β-ketoacyl-ACP synthases I (fabB from E. coli),Enzyme: β-ketoacyl-ACP synthases II (fabF from E. coli), Enzyme:malonyl-CoA-ACP transacylase (fabD from E. coli), Enzyme: pantothenatekinase (coaA from E. coli), Enzyme: pyruvate dehydrogenase complex(aceEF from E. coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase(mmsB from Pseudomonas aeruginosa), Enzyme: lipoamide dehydrogenase (lpdfrom E. coli), Enzyme: γ-glutamyl-γ-aminobutyraldehyde dehydrogenase(puuC from E. coli), Enzyme: malate synthase A (aceB from E. coli),Enzyme: isocitrate lyase (aceA from E. coli), Enzyme: isocitratedehydrogenase phosphatase/kinase (aceK from E. coli), Enzyme: 3-hydroxyacid dehydrogenase (ydfG from E. coli), Enzyme: acetyl CoA carboxylase(accADBC from E. coli), Polypeptide: predicted transcriptional regulator(yieP from E. coli), Blastocyin resistance gene (BSD fromSchizosaccharomyces pombe), Enzyme: pyridine nucleotide transhydrogenase(udha from E. coli), Protein: Cra DNA-binding transcriptional dualregulator (fruR from E. coli), (SCB from E. coli), enzyme: aldehydedehydrogenase B (aldB from E. coli), Enzyme: carbonic anhydrase (cynTfrom E. coli), Enzyme: cyanase (cynS from E. coli), DNA gyrasetoxin-antitoxin system (ccdAB from E. coli), Enzyme: phosphoglyceratemutase (pgi from E. coli), ArcA transcriptional dual regulator orAerobic respiration control (arcA from E. coli), Enzyme:6-phosphofructokinase (pfk from E. coli), Enzyme: glyceraldehyde3-phosphate dehydrogenase-A complex (gapA from E. coli), aldehydedehydrogenase A (alda from E. coli), Enzyme: glutamate dehydrogenase(gdhA from E. coli), Enzyme: NADH-dependent serine dehydrogenase (NDSDfrom Pseudomonas aeruginosa), Protein: threonine/homoserine effluxtransporter (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphatedehydrogenase (gapN from E. coli), Phosphotransferase system (pts fromE. coli), Enzyme: 6-phosphofructokinase II (pfkB from E. coli), Enzyme:methylmalonate-semialdehyde dehydrogenase (mmsA from Pseudomonasaeruginosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 fromBacillus cereus), Enzyme: aspartate 1-decarboxylase (panD from E. coli),Gene that confers resistance to valine (ValR from E. coli), Enzyme:glucokinase (glk from E. coli), Polypeptide: 30 S ribosomal sununitprotein S12 (rpsL from E. coli), Polypeptide: CynR DNA-bindingtranscriptional repressor (cynR from E. coli), Transporter: galactose:H+symporter (galP from E. coli), aspartate aminotransferase (aspC from E.coli), Enzyme: alpha-ketoglutarate reductase (serA from E. coli),Enzyme: 6-phosphofructokinase I (pfkA from E. coli), Enzyme:phosphoenolpyruvate carboxylase (ppc from E. coli), Enzyme:succinate-semialdehyde dehydrogenase (NADP+) (gabD from E. coli),Enzyme: pyruvate kinase (pyk from E. coli), Enzyme: oxaloacetate4-decarboxylase (OAD from Leuconostoc mesenteroides), Enzyme: triggerfactor; a molecular chaperone involved in cell division (tig from E.coli), Transcription Unit (ptsHIcrr from E. coli), Enzyme: acetyl-CoAacetaldehyde dehydrogenase/alcohol dehydrogenase (adhE from E. coli),Enzyme: fattyacyl thioesterase I (tesA from E. coli), Enzyme: guanosine3′-diphosphate 5′-triphosphate 3′-diphosphatase (spoT from E. coli),combination of genes encoding accABCD subunits (from E. coli andHalomonas elongata), pol (from E. coli), Enzyme: GDPpyrophosphokinase/GTP pyrophosphokinase (relA from E. coli), [EnzymeName] (me from E. coli), Enzyme: citrate synthase (gltA from E. coli),Polypeptide: DNA gyrase, subunit A (gyrA from E. coli), Enzyme:multifunctional 2-keto-3-deoxygluconate 6-phosphate aldolase and2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (edafrom E. coli), thiamin biosynthesis (thi from E. coli), Polypeptide:acetolactate synthase II (ilvG from E. coli), acetyl CoA carboxylase(accDACB from E. coli), Citrate synthase (ArCS from Arthrobacteraurescens), Acetyl-CoA carboxylase from Corynebacter glutamicum (CgACCfrom Corynebacter glutamicum), Polypeptide: ferrichrome/phage/antibioticouter membrane porin FhuA (fhuA from E. coli), Transporter: phosphate:H+symporter PitA (pitA from E. coli), Transporter: uracil:H+ symporter(uraA from E. coli), Enzyme: uracil phosphoribosyltransferase (upp fromE. coli), Enzyme: acylphosphatase (yccX from E. coli), acetyl-CoAsynthetase (acsA from E. coli), Polypeptide: restriction of methylatedadenine (mrr from E. coli), Protein: TrpR transcriptional repressor(trpR from E. coli), Enzymes: glutamate 5-semialdehydedehydrogenase/gamma-glutamyl kinase (proAB from E. coli), methylcytosinerestriction system (mcrBC from E. coli), Protein: citrate lyase,citrate-ACP transferase component (citF from E. coli), Enzyme:thioesterase II (tesB from E. coli), Enzyme: DNA-specific endonuclease I(endA from E. coli), Enzyme: phosphate acetyltransferase (eutD from E.coli), Enzyme: propionate kinase (tdcD from E. coli), tRNA: tRNA glnV(supE from E. coli), Enzyme: DNA-binding, ATP-dependent protease La (lonfrom E. coli), Polypeptide: DNA strand exchange and recombinationprotein with protease and nuclease activity (recA from E. coli),Transcription Unit: restriction endonulease component of EcoKIrestriction-modification system (hsdRMS from E. coli), Enzyme:restriction of DNA at 5-methylcytosine residues (mcrA from E. coli). Insome embodiment the genetic modifications listed above are modifiedfurther with the genetic modules provided herein.

In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the method of bioproduction ofvarious chemicals which can be used to make various consumer productsdescribed herein.

In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the bioproduction of 1,4-butanediol(1,4-BDO) (U.S. Pub. No. 20110190513). In some embodiment the geneticmodification of the genes, proteins and enzymes of the invention can befor the bioproduction of butanol (U.S. application Ser. No. 13/057,359).In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the bioproduction of isobutanol(U.S. application Ser. No. 13/057,359).

In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the bioproduction of 3-HP such andits aldehyde metabolites (U.S. application Ser. No. 13/062,917).

In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the bioproduction of polyketidechemical products (U.S. application Ser. No. 13/575,581).

In some embodiment the genetic modification of the genes, proteins andenzymes of the invention can be for the bioproduction of fatty acidmethyl esters (U.S. Pub. No. 20110124063). In some embodiment thegenetic modification of the genes, proteins and enzymes of the inventioncan be for the bioproduction of C4-C18 fatty acids (U.S. ApplicationSer. No. 61/682,127).

Genetic Modifications

Various methods to achieve such genetic modification in a host strainare well known to one skilled in the art. Example of geneticmodification that can be used by the claimed invention include, but arenot limited to, increasing expression of an endogenous genetic element;increasing expression of an exogenous genetic element; decreasingfunctionality of a repressor gene; increasing functionality of arepressor gene; increasing functionality of a activator gene; decreasingfunctionality of a activator gene; introducing a genetic change orelement integrated in the host genome, introducing a heterologousgenetic element permanently, by integration into the genome ortransiently by transformation with plasmid; increasing copy number of anucleic acid sequence encoding a polypeptide catalyzing an enzymaticconversion step; mutating a genetic element to provide a mutated proteinto increase specific enzymatic activity; mutating a genetic element toprovide a mutated protein to decrease specific enzymatic activity;over-expressing of gene; reduced the expression of a gene; knocking outor deleting a gene; altering or modifying feedback inhibition; providingan enzyme variant comprising one or more of an impaired binding sites oractive sites; increasing functionality of a siRNA, decreasingfunctionality of a siRNA, increasing functionality of a antisensemolecule, decreasing functionality of a antisense molecule, addition ofgenetic modules such as RBS, '3 UTR elements to increase mRNA stabilityor translation; deletion of genetic modules such as RBS, '3 UTR elementsto decrease mRNA stability or translation; addition or modification ofgenetic modules such as '5 UTR elements to increase transcription;deletion or modification of genetic modules such as '5 UTR and elementsto increase transcription. In addition other genetic modules, provideherein, such a multicopy plasmids and various promoters can be used tofurther modify of the genetic modifications provide herein.Additionally, as known to those of ordinarily skill in the art compoundssuch as siRNA technology, anti-sense technology, and small molecule ininhibitors can be used to alter gene expression in the same manner as agenetic modification.

Screening methods, such as SCALE in combination with random mutagenesismay be practiced to provide genetic modifications that provide a benefitto increased production of 3-HP in a host cell. Examples of randommutagenesis can include insertions, deletions and substitutions of oneor more nucleic acids in a nucleic acid of interest. In variousembodiments a genetic modification results in improved enzymaticspecific activity and/or turnover number of an enzyme. Without beinglimited, changes may be measured by one or more of the following: KM;Kcat, Kavidity, gene expression level, protein expression level, levelof a product known to be produced by the enzyme, 3-HP tolerance, or by3-HP production or by other means.

Host Cells

In some applications of the invention the host cell can be agram-negative bacterium. In some applications of the invention the hostcell can be from the genera Zymomonas, Escherichia, Pseudomonas,Alcaligenes, or Klebsiella. In some applications of the invention thehost cell can be Escherichia coli, Cupriavidus necator, Oligotrophacarboxidovorans, or Pseudomonas putida. In some applications of theinvention the host cell is one or more an E. coli strains.

In some applications of the invention the host cell can be agram-positive bacterium. In some applications of the invention the hostcell can be from the genera Clostridium, Salmonella, Rhodococcus,Bacillus, Lactobacillus, Enterococcus, Paenibacillus, Arthrobacter,Corynebacterium, or Brevibacterium. In some applications of theinvention the host cell is Bacillus licheniformis, Paenibacillusmacerans, Rhodococcus erythropolis, Lactobacillus plantarum,Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis,or Bacillus subtilis. In some applications of the invention the hostcell is B. subtilis strain.

In some applications of the invention the host cell is yeast. In someapplications of the invention the host cell can be from the generaPichia, Candida, Hansenula or Saccharomyces. In some applications of theinvention the host cell is Saccharomyces cerevisiae. In someapplications of the invention the host cell is Saccharomyces pombe.

In some applications of the invention the host cell is an alga. In someapplications of the invention the host cell is a halophile. In someapplications of the invention the host cell is an alga. In someapplications of the invention the host cell is a chemolithotrophicbacterium.

In some applications of the invention the host cell is comprised ofmultiple host cell types. In some applications of the invention the hostcell is comprised of one host cell type. In some applications of theinvention the host cell is comprised of one more species or strain of ahost cell type.

Downstream Consumer Products Chemicals

3-HP purified according to the methods provided in this disclosure maybe converted to various other products having industrial uses including,but not limited to, acrylamide, acrylic acid, esters of acrylic acid,1,3-propanediol, and other chemicals, collectively referred to as“downstream chemical products” or “downstream products.” In someinstances the conversion is associated with the separation and/orpurification steps. These downstream chemical products are useful forproducing a variety of consumer products which are described in moredetail below. The methods of the present invention include steps toproduce downstream products of 3-HP.

As a C3 building block, 3-HP offers much potential in a variety ofchemical conversions to commercially important intermediates, industrialend products, and consumer products. For example, 3-HP may be convertedto acrylic acid, acrylates (e.g., acrylic acid salts and esters),1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxypropionate, propiolactone, acrylamide, or acrylonitrile.

Additionally, 3-HP may be oligomerized or polymerized to formpoly(3-hydroxypropionate) homopolymers, or co-polymerized with one ormore other monomers to form various co-polymers. Because 3-HP has asingle stereoisomer, polymerization of 3-HP is not complicated by thestereo-specificity of monomers during chain growth. This is in contrastto (S)-2-hydroxypropanoic acid (also known as lactic acid), which hastwo (D, L) stereoisomers that should be considered during itspolymerizations.

As will be further described, 3-HP can be converted into derivativesstarting (i) substantially as the protonated form of 3-hydroxypropionicacid; (ii) substantially as the deprotonated form, 3-hydroxypropionate;or (iii) as mixtures of the protonated and deprotonated forms.Generally, the fraction of 3-HP present as the acid versus the salt willdepend on the pH, the presence of other ionic species in solution,temperature (which changes the equilibrium constant relating the acidand salt forms), and, to some extent, pressure. Many chemicalconversions may be carried out from either of the 3-HP forms, andoverall process economics will typically dictate the form of 3-HP fordownstream conversion.

Acrylic acid obtained from 3-HP purified by the methods described inthis disclosure may be further converted to various polymers. Forexample, the free-radical polymerization of acrylic acid takes place bypolymerization methods known to the skilled worker and can be carriedout, for example, in an emulsion or suspension in aqueous solution oranother solvent. Initiators, such as but not limited to organicperoxides, are often added to aid in the polymerization. Among theclasses of organic peroxides that may be used as initiators are diacyls,peroxydicarbonates, monoperoxycarbonates, peroxyketals, peroxyesters,dialkyls, and hydroperoxides. Another class of initiators is azoinitiators, which may be used for acrylate polymerization as well asco-polymerization with other monomers. U.S. Pat. Nos. 5,470,928;5,510,307; 6,709,919; and 7,678,869 teach various approaches topolymerization using a number of initiators, including organicperoxides, azo compounds, and other chemical types, and are incorporatedby reference for such teachings as applicable to the polymers describedherein.

Accordingly, it is further possible for co-monomers, such ascrosslinkers, to be present during the polymerization. The free-radicalpolymerization of the acrylic acid obtained from dehydration of 3-HP, asproduced herein, in at least partly neutralized form and in the presenceof crosslinkers is practiced in certain embodiments. This polymerizationmay result in hydrogels which can then be comminuted, ground and, whereappropriate, surface-modified, by known techniques.

An important commercial use of polyacrylic acid is for superabsorbentpolymers. This specification hereby incorporates by reference ModernSuperabsorbent Polymer Technology, Buchholz and Graham (Editors),Wiley-VCH, 1997, in its entirety for its teachings regardingsuperabsorbent polymers components, manufacture, properties and uses.Superabsorbent polymers are primarily used as absorbents for water andaqueous solutions for diapers, adult incontinence products, femininehygiene products, and similar consumer products. In such consumerproducts, superabsorbent materials can replace traditional absorbentmaterials such as cloth, cotton, paper wadding, and cellulose fiber.Superabsorbent polymers absorb, and retain under a slight mechanicalpressure, up to 25 times or more their weight in liquid. The swollen gelholds the liquid in a solid, rubbery state and prevents the liquid fromleaking Superabsorbent polymer particles can be surface-modified toproduce a shell structure with the shell being more highly cross-linkedthan the rest of the particle. This technique improves the balance ofabsorption, absorption under load, and resistance to gel-blocking. It isrecognized that superabsorbent polymers have uses in fields other thanconsumer products, including agriculture, horticulture, and medicine.

Superabsorbent polymers are prepared from acrylic acid (such as acrylicacid derived from 3-HP provided herein) and a crosslinker, by solutionor suspension polymerization. Exemplary methods include those providedin U.S. Pat. Nos. 5,145,906; 5,350,799; 5,342,899; 4,857,610; 4,985,518;4,708, 997; 5,180,798; 4,666,983; 4,734,478; and 5,331,059, eachincorporated by reference for their teachings relating to superabsorbentpolymers.

Among consumer products, a diaper, a feminine hygiene product, and anadult incontinence product are made with superabsorbent polymer thatitself is made substantially from acrylic acid converted from 3-HP madein accordance with the present invention.

Diapers and other personal hygiene products may be produced thatincorporate superabsorbent polymers made from acrylic acid made from3-HP which is produced and purified by the teachings of the presentapplication. The following provides general guidance for making a diaperthat incorporates such superabsorbent polymer. The superabsorbentpolymer first is molded into an absorbent pad that may be vacuum formed,and in which other materials, such as a fibrous material (e.g., woodpulp) are added. The absorbent pad then is assembled with sheet(s) offabric, generally a nonwoven fabric (e.g., made from one or more ofnylon, polyester, polyethylene, and polypropylene plastics) to formdiapers.

More particularly, in one non-limiting process, multiple pressurizednozzles, located above a conveyer belt, spray superabsorbent polymerparticles (e.g., about 400 micron size or larger), fibrous material,and/or a combination of these onto the conveyer belt at designatedspaces/intervals. The conveyor belt is perforated and under vacuum frombelow, so that the sprayed on materials are pulled toward the beltsurface to form a flat pad. In various embodiments, fibrous material isapplied first on the belt, followed by a mixture of fibrous material andthe superabsorbent polymer particles, followed by fibrous material, sothat the superabsorbent polymer is concentrated in the middle of thepad. A leveling roller may be used toward the end of the belt path toyield pads of uniform thickness. Each pad thereafter may be furtherprocessed, such as to cut it to a proper shape for the diaper, or thepad may be in the form of a long roll sufficient for multiple diapers.Thereafter, the pad is sandwiched between a top sheet and a bottom sheetof fabric (one generally being liquid pervious, the other liquidimpervious), for example on a conveyor belt, and these are attachedtogether, for example by gluing, heating or ultrasonic welding, and cutinto diaper-sized units (if not previously so cut). Additional featuresmay be provided, such as elastic components, strips of tape, etc., forfit and ease of wearing by a person.

The ratio of the fibrous material to polymer particles is known toaffect performance characteristics. In some cases, this ratio is between75:25 and 90:10 (see e.g., U.S. Pat. No. 4,685,915, incorporated byreference for its teachings of diaper manufacture). Other disposableabsorbent articles may be constructed in a similar fashion, such asabsorbent articles for adult incontinence, feminine hygiene (sanitarynapkins), tampons, etc. (see, for example, U.S. Pat. Nos. 5,009,653;5,558,656; and 5,827,255 incorporated by reference for their teachingsof sanitary napkin manufacture).

Low molecular weight polyacrylic acid has uses for water treatment, andas a flocculant and thickener for various applications includingcosmetics and pharmaceutical preparations. For these applications, thepolymer may be uncrosslinked or lightly cross- linked, depending on thespecific application. The molecular weights are typically from about 200to about 1,000,000 g/mol. Preparation of these low molecular weightpolyacrylic acid polymers is described in U.S. Pat. Nos. 3,904,685;4,301,266; 2,798,053; and 5,093,472, each of which is incorporated byreference for its teachings relating to methods to produce thesepolymers.

Acrylic acid may be co-polymerized with one or more other monomersselected from acrylamide, 2-acrylamido-2-methylpropanesulfonic acid,N,N-dimethylacrylamide, N-isopropylacrylamide, methacrylic acid, andmethacrylamide, to name a few. The relative reactivities of the monomersaffect the microstructure and thus the physical properties of thepolymer. Co-monomers may be derived from 3-HP, or otherwise provided, toproduce co-polymers. Ullmann's Encyclopedia of Industrial Chemistry,Polyacrylamides and Poly(Acrylic Acids), Wi1eyVCH Verlag GmbH, Wienham(2005), is incorporated by reference herein for its teachings of polymerand co-polymer processing.

Acrylic acid can in principle be copolymerized with almost anyfree-radically polymerizable monomers including styrene, butadiene,acrylonitrile, acrylic esters, maleic acid, maleic anhydride, vinylchloride, acrylamide, itaconic acid, and so on. End-use applicationstypically dictate the co-polymer composition, which influencesproperties. Acrylic acid also may have a number of optionalsubstitutions and, after such substitutions, may be used as a monomerfor polymerization, or co-polymerization reactions. As a general rule,acrylic acid (or one of its co-polymerization monomers) may besubstituted by any substituent that does not interfere with thepolymerization process, such as alkyl, alkoxy, aryl, heteroaryl, benzyl,vinyl, allyl, hydroxy, epoxy, amide, ethers, esters, ketones,maleimides, succinimides, sulfoxides, glycidyl and silyl (see e.g., U.S.Pat. No. 7,678,869, incorporated by reference above, for furtherdiscussion). The following paragraphs provide a few non- limitingexamples of copolymerization applications.

Paints that comprise polymers and copolymers of acrylic acid and itsesters are in wide use as industrial and consumer products. Aspects ofthe technology for making such paints can be found in e.g., U.S. Pat.Nos. 3,687,885 and 3,891,591, incorporated by reference for theirteachings of such paint manufacture. Generally, acrylic acid and itsesters may form homopolymers or copolymers among themselves or withother monomers, such as amides, methacrylates, acrylonitrile, vinyl,styrene and butadiene. A desired mixture of homopolymers and/orcopolymers, referred to in the paint industry as “vehicle” (or “binder”)are added to an aqueous solution and agitated sufficiently to form anaqueous dispersion that includes sub-micrometer sized polymer particles.The paint cures by coalescence of these vehicle particles as the waterand any other solvent evaporate. Other additives to the aqueousdispersion may include pigment, filler (e.g., calcium carbonate,aluminum silicate), solvent (e.g., acetone, benzol, alcohols, etc.,although these are not found in certain no VOC paints), thickener, andadditional additives depending on the conditions, applications, intendedsurfaces, etc. In many paints, the weight percent of the vehicle portionmay range from about nine to about 26 percent, but for other paints theweight percent may vary beyond this range.

Acrylic-based polymers are used for many coatings in addition to paints.For example, for paper coating latexes, acrylic acid is used from0.1-5.0%, along with styrene and butadiene, to enhance binding to thepaper and modify rheology, freeze-thaw stability and shear stability. Inthis context, U.S. Pat. Nos. 3,875,101 and 3,872,037 are incorporated byreference for their teachings regarding such latexes. Acrylate-basedpolymers also are used in many inks, particularly UV curable printinginks For water treatment, acrylamide and/or hydroxy ethyl acrylate arecommonly co-polymerized with acrylic acid to produce lowmolecular-weight linear polymers. In this context, U.S. Pat. Nos.4,431,547 and 4,029,577 are incorporated by reference for theirteachings of such polymers. Co-polymers of acrylic acid with maleic acidor itaconic acid are also produced for water-treatment applications, asdescribed in U.S. Pat. No. 5,135,677, incorporated by reference for thatteaching. Sodium acrylate (the sodium salt of glacial acrylic acid) canbe co-polymerized with acrylamide (which may be derived from acrylicacid via amidation chemistry) to make an anionic co- polymer that isused as a flocculant in water treatment.

For thickening agents, a variety of co-monomers can be used, such asthose described in U.S. Pat. Nos. 4,268,641 and 3,915,921, incorporatedby reference for their description of these co-monomers. U.S. Pat. No.5,135,677 describes a number of co-monomers that can be used withacrylic acid to produce water-soluble polymers, and is incorporated byreference for such description.

In some cases, conversion to downstream products may be madeenzymatically. For example, 3-HP may be converted to 3-HP-CoA, whichthen may be converted into polymerized 3-HP with an enzyme havingpolyhydroxy acid synthase activity (EC 2.3.1.-). Also, 1,3-propanediolcan be made using polypeptides having oxidoreductase activity orreductase activity (e.g., enzymes in the EC 1.1.1.-class of enzymes).Alternatively, when creating 1,3-propanediol from 3-HP, a combination of(1) a polypeptide having aldehyde dehydrogenase activity (e.g., anenzyme from the 1.1.1.34 class) and (2) a polypeptide having alcoholdehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can beused. Polypeptides having lipase activity may be used to form esters.Enzymatic reactions such as these may be conducted in vitro, such asusing cell-free extracts, or in vivo.

Thus, various embodiments described in this disclosure, such as methodsof making a chemical, include conversion steps to any downstreamproducts of microbially produced 3-HP, including but not limited tothose chemicals described herein, in the incorporated references, andknown in the art. For example, in some cases, 3-HP is produced andconverted to polymerized-3-HP (poly-3-HP) or acrylic acid. In somecases, 3-HP or acrylic acid can be used to produce polyacrylic acid(polymerized acrylic acid, in various forms), methyl acrylate,acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid,1,3-propanediol, ethyl acrylate, n-butyl acrylate, hydroxypropylacrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexylacrylate, and acrylic acid or an acrylic acid ester to which an alkyl oraryl addition may be made, and/or to which halogens, aromatic amines oramides, and aromatic hydrocarbons may be added.

-   -   a) Reactions that form downstream compounds such as acrylates or        acrylamides can be conducted in conjunction with use of suitable        stabilizing agents or inhibiting agents reducing the likelihood        of polymer formation. See, for example, U.S. Publication No.        2007/0219390, incorporated by reference in its entirety.        Stabilizing agents and/or inhibiting agents include, but are not        limited to, e.g., phenolic compounds (e.g., dimethoxyphenol        (DMP) or alkylated phenolic compounds such as di-tert-butyl        phenol), quinones (e.g., t-butyl hydroquinone or the monomethyl        ether of hydroquinone (MEHQ)), and/or metallic copper or copper        salts (e.g., copper sulfate, copper chloride, or copper acetate)        Inhibitors and/or stabilizers can be used individually or in        combinations as will be known by those of skill in the art.

In some cases, the one or more downstream compounds are recovered at amolar yield of up to about 100 percent, or a molar yield in the rangefrom about 70 percent to about 90 percent, or a molar yield in the rangefrom about 80 percent to about 100 percent, or a molar yield in therange from about 90 percent to about 100 percent. Such yields may be theresult of single-pass (batch or continuous) or iterative separation andpurification steps in a particular process.

The methods described in this disclosure can also be used to producedownstream compounds derived from 3-HP, such as but not limited to,polymerized-3-HP (poly-3-HP), acrylic acid, polyacrylic acid(polymerized acrylic acid, in various forms), copolymers of acrylic acidand acrylic esters, acrylamide, acrylonitrile, propiolactone, ethyl3-HP, malonic acid, and 1,3-propanediol. Also, among esters that areformed are methyl acrylate, ethyl acrylate, n-butyl acrylate,hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, and2-ethylhexyl acrylate. These and/or other acrylic acid and/or otheracrylate esters may be combined, including with other compounds, to formvarious known acrylic acid-based polymers. Numerous approaches may beemployed for such downstream conversions, generally falling intoenzymatic, catalytic (chemical conversion process using a catalyst),thermal, and combinations thereof (including some wherein a desiredpressure is applied to accelerate a reaction). For example, withoutbeing limiting, acrylic acid may be made from 3-HP via a dehydrationreaction, methyl acrylate may be made from 3-HP via dehydration andesterification, the latter to add a methyl group (such as usingmethanol), acrylamide may be made from 3-HP via dehydration andamidation reactions, acrylonitrile may be made via a dehydrationreaction and forming a nitrile moiety, propiolactone may be made from3-HP via a ring-forming internal esterification reaction, ethyl-3-HP maybe made from 3-HP via esterification with ethanol, malonic acid may bemade from 3-HP via an oxidation reaction, and 1,3-propanediol may bemade from 3-HP via a reduction reaction. Additionally, it is appreciatedthat various derivatives of the derivatives of 3-HP and acrylic acid maybe made, such as the various known polymers of acrylic acid and itsderivatives. Production of such polymers is considered within the scopeof the present invention. Copolymers containing acrylic acid and/oresters have been widely used in the pharmaceutical formulation toachieve extended or sustained release of active ingredients, for exampleas coating material. Downstream compounds may also be converted toconsumer products such as diapers, carpet, paint, and adhesives.

Another important product, acrylamide, has been used in a number ofindustrial applications. Acrylamide may be produced from 3-HP, forexample, without being limiting, via anesterification-amidation-dehydration sequence. Refluxing an alcoholsolution of 3-HP in the presence of an acid or Lewis acid catalystdescribed herein would lead to a 3-HP ester. Treatment of the 3-HP esterwith either an ammonia gas or an ammonium ion could yield 3-HP amide.Finally, dehydration of the 3-HP amide with dehydration reagentsdescribed elsewhere in this disclosure could produce acrylamide. Thesteps mentioned herein may be rearranged to produce the same finalproduct acrylamide. Polymerization of acrylamide can be achieved, forexample, and without being limiting, by radical polymerization.Polyacrylamide polymers have been widely used as additives for treatingmunicipal drinking water and waste water. In addition, they have foundapplications in gel electrophoresis, oil- drilling, papermaking, oreprocessing, and the manufacture of permanent press fabrics.

-   -   VIII. EXPRESSION SYSTEMS GENERAL CONCEPTS

The following general concepts are applicable to embodiments of theinvention described above.

Multicopy Plasmids

The researcher is faced with a myriad of genetic module options whendesigning a plasmid for expression of a heterologous protein in a hostcell. How to optimize an expression plasmid system often depends on thedownstream use of the expressed protein.

Different cloning vectors or plasmids are maintained at different copynumbers, dependent on the replicon of the plasmid. Most general cloningplasmids can carry a DNA insert up to around 15 kb in size.

Multicopy plasmids can be used for the expression of recombinant genesin Escherichia coli. Examples of include multicopy plasmids includehigh-copy, medium-copy and low-copy plasmids (see FIG. 8). The high copynumber is generally desired for maximum gene expression. However, themetabolic burden effects can result from multiple plasmid copies couldprove to be detrimental for maximum productivity in certain metabolicengineering applications by adding significant metabolic burden to thesystem.

The low-copy plasmids for example, pBR322 is based on the original ColE1replicon and thus has a copy number of 15-20. The pACYC series ofplasmids are based on the p15A replicon, which has a copy number of18-22, whereas pSC101 has even a lower copy number around 5, and BACsare maintained at one copy per cell. Such low copy plasmids may beuseful in metabolic engineering applications, particularly when one ormore of the substrates used in the recombinant pathway are required fornormal cellular metabolism and can be toxic to the cell at high levels.

However, the used of high-copy plasmids may be useful in enhancedcellular metabolism contexts. The mutant ColE1 replicon, as found in thepUC series of plasmids produces a copy number of 500-700 as a result ofa point mutation within the RNAII regulatory molecule.

There are transcription and translation vectors. Transcription vectorsare utilized when the DNA to be cloned has an ATG start codon and aprokaryotic ribosome-binding site. Translation vectors contain anefficient ribosome-binding site and, therefore, it is not necessary forthe target DNA to contain one. This is particularly useful in caseswhere the initial portion of the gene may be cleaved in an effort toimprove solubility. Another consideration when choosing a transcriptionor translation vector is the source of the DNA to be expressed.Prokaryotic genes usually have a ribosome-binding site that iscompatible with the host E. coli translation machinery, whereaseukaryotic genes do not. Normal prokaryotic gene expression may beenhanced by use of an engineered promoter and ribosome-binding site.

Promoters

A promoter is a region of DNA that initiates transcription of aparticular gene. In bacteria, transcription is initiated by the promoterbeing recognized by RNA polymerase and an associated sigma factor, whichare often brought to the promoter site by an activator protein's bindingto its own DNA binding site located by the promoter.

Promoter selection is an important factor when designing an expressionplasmid system. A promoter is located upstream of the ribosome-bindingsite. Owing to the fact that many heterologous protein products aretoxic to the cell, the promoter can be regulated so that theheterologous protein is expressed at the appropriate amount and time toreduced the burden on the cell host.

Historically, the most commonly used promoters have been the lactose(lac) and tryptophan (trp) promoters. These two promoters were combinedto create the hybrid promoters tac and trc that are also commonly used.Other common promoters are the phage lambda promoters, the phage T7promoter (T7), and the alkaline phosphatase promoter (phoA).

Promoters can be constitutive and inducible. Constitutive promoter isactive in all circumstances in the cell, while regulated or induciblepromoter become active in response to specific stimuli. In addition thestrength of the promoter can also differ. A strong promoter has a highfrequency of transcription and generates the heterologous protein as10-30% of the total cellular protein production (for examples see FIG.8). Chemically-inducible promoters that can be used in various aspectsof the invention include but are not limited to promoters whosetranscriptional activity is regulated by the presence or absence ofalcohol, tetracycline, steroids, metal and other compounds.Physically-inducible promoters that can be used in various aspects ofthe invention include but are not limited to including promoters whosetranscriptional activity is regulated by the presence or absence oflight and low or high temperatures.

In order to be an inducible promoter, the promoter should be initiallybe completely repressed to transcription and then transcription inducedwith the addition of an inducer to allow expression at the time thatexpression is desired in the host cell. Alternatively, an induciblepromoter may be responsive to the lack of a substance, such as inorganicphosphate, such that the absence of inorganic phosphate will allowexpression at the time that expression is desired in the host cell (forexamples see FIG. 8).

Ribosome Binding Sites

A Ribosome Binding Sites (RBS) is an RNA sequence upstream of the startcodon that affects the rate at which a particular gene or open readingframe (ORF) is translated. One can tailor an RBS site to a particulargene. Ribosome Binding Sites (RBSs) are typically short sequences, oftenless than 20 base pairs. Various aspects of RBS design are known toaffect the rate at which the gene is translated in the cell. The RBSmodule can influences the translation rate of a gene largely by twoknown mechanisms. First, the rate at which ribosomes are recruited tothe mRNA and initiate translation is dependent on the sequence of theRBS. Secondly, the sequence of the RBS can also affect the stability ofthe mRNA in the cell, which in turn affects the number of proteins.Through the use of genetic expression modules the expression of desiredgenes, such as genes encoding enzymes in the biosynthetic pathway for3-HP, can be tailored activity either at the transcriptional andtranslational level.

One can access the registry RBS collection as a starting point fordesigning an RBS<<http://partsregistry.org/Ribosome_Binding_Sites/Catalog>>. TheRegistry has collections of RBSs that are recommended for generalprotein expression in E. coli and other prokaryotic hosts. In addition,each family of RBSs has multiple members covering a range of translationinitiation rates. There are also several consensus RBS sequence for E.coli have been described. However, it is important to keep in mind theknown RBS functions and mechanisms in a larger context. For example, incertain contexts the RBS can interact with upstream sequences, such assequence that comprise the promoter or an upstream ORF. In othercontexts, the RBS may interact with downstream sequences, for examplethe ribosome enzyme binds jointly to the RBS and start codon at aboutthe same time. These potential interactions should be considered in theoverall RBS sequence design. The degree of secondary structure near theRBS can affect the translation initiation rate. This fact can be used toproduce regulated translation initiation rates.

The Shine-Dalgarno portion of the RBS is critical to the strength of theRBS. The Shine-Dalgarno is found at the end of the 16s rRNA and is theportion that binds with the mRNA and includes the sequence 5′-ACCUCC-3′.RBSs will commonly include a portion of the Shine-Dalgarno sequence. Oneof the ribosomal proteins, S1, is known to bind to adenine basesupstream from the Shine-Dalgarno sequence. As a result, the RBS can bemade stronger by adding more adenines in the sequence upstream of theRBS.

When considering the design of the spacing between the RBS and the startcodon, it is important to think of the aligned spacing rather than justthe absolute spacing. While the Shine-Dalgarno portion of the RBS iscritical to the strength of the RBS, the sequence upstream of theShine-Dalgarno sequence is also important. Note that the promoter mayadd some bases onto the start of the mRNA that may affect the strengthof the RBS by affecting S1 binding.

Computer programs that design RBS sequence to match protein codingsequences, desired upstream sequences including regulatory mRNAsequences, and account of secondary structure are known [Salis, Mirsky,and Voight, Nature Biotechnology 27: 946-950, 2009] and were used tooptimize RBSs for the ACCase subunit genes as described in (see EXAMPLE3).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1: Salt Inhibition Studies in E. Coli

The activity of ACCase complex, a critical enzyme in the conversion ofacetyl-CoA to malonyl-CoA, the immediate precursor for 3-HP, is severelyinhibited by salt. Dose-dependent effects on ACCase activity wasobserved in the presence of NaCl, NH₄Cl, Na-3-HP, or NH₄-3-HP such thatsalt levels near 0.44 M resulted in decreasing the activity of theACCase enzyme by approximately 80%, while salts of 3-HP levels near 0.66M decreased the activity of the ACCase enzyme by approximately 80%relative to control (FIG. 4). Levels of greater than 0.66 M (60 g/L) areexpected to be present for commercially viable commercial production of3-HP.

Example 2: ACCase from Halophilic Organism

Halophilic organisms, such as Halomonas elongata, are found inenvironments with high salt concentrations and, in general, have a saltinternal concentration >2.5-3 M. It is hypothesized that enzymes derivedfrom any salt-tolerant species should be more resistant to enzymeinhibition by salts, such as 3-HP. Further, these enzymes that havegreater salt tolerance should in turn have extended production underhigh salt conditions than enzymes with lower salt tolerance.

Accordingly, the genes encoding the accA, accB, accC, accD of H.elongata described in Table 1 were synthesized for expression in E. coliusing codons optimized for this organism and supplied individually onpUC57 plasmids without promoters. Synthetic operons comprising thesubunits were assembled using the Gibson assembly method.

TABLE 1 Accession numbers for genes encoding ACCase subunits fromHalomonas elongata Gene Accession number SEQ ID NO. accA YP_003898857.1SEQ ID NO. 1, 2 accB YP_003897250.1 SEQ ID NO. 3, 4 accC YP_003897249.1SEQ ID NO. 5, 6 accD YP_003897309.1 SEQ ID NO. 7, 8Each gene was amplified by PCRs with Pfu Ultra II HS using themanufacturer's instructions, and the PCR products were purified usingthe Zymo PCR Cleanup kit. Concentrations of products were measured usingthe Nanodrop spectophotometer. The Gibson Assembly kit (NEB) was used toconstruct plasmids expressing the ACCase subunit genes as directed bythe manufacturer. The effect of NH₄-3-HP and NH₄Cl on H. elongata ACCasewas tested and compared to the E. coli ACCase. As shown in FIG. 4,whereas the E. coli ACCase is significantly inhibited by the salts, theACCase from the halophile is less affected by either NH₄-3-HP or byNH₄Cl. This result indicated that use of the H. elongata ACCase in 3-HPproduction strains would in beneficial in relieving 3-HP inhibition ofthe conversion of acetyl-CoA to malonyl-CoA, a critical step in thepathway.

Example 3: RBS-Optimized Genes

Enzyme expression is regulated at transcriptional and translationallevels in prokaryotes. Ribosome Binding Sites (RBS) are 15 nucleotidesegments which are known to control the level of protein expression inmicroorganisms. To enhance H. elongata ACCase expression variouscustomized RBS were constructed and optimized for E. coli translationexpression. Table 2 shows the RBS sequences used to increase expressionof the individual subunits.

TABLE 2 Table 2 RBS sequences used to enhance expression ofH. elongate ACCase subunits. H. elongata ACC expressionModified RBS sequences preceeding ATG (underlined) plasmid He_accDHe_accA He_accC He_accB Parent 2-4 5′- 5′- 5′- 5′- GCGTAGTAAAGGACAATTTATTTAAGGA GAAATTTCATACC GGAAGAACAAGGG GGTAACATATG GGACTCTTAAGATGACAGGCGAAGGAG GTGTACATG GAAAAACCATG B2 Same as 2-4 Same as 2-4Same as 2-4 5′- ggaagaattaagg gggacaaggggga ataATG 13A 5′- Same as 2-4Same as 2-4 gcgtagtagccggg tgataaggagccgt aacATG 14C 5′- Same as 2-4Same as 2-4 Same as 2-4 gcgtagtagctgat ataaaaggaggtaa cggATG 15CSame as 2-4 5′- Same as 2-4 Same as 2-4 caatttatttttgtt cacccaaggagtattgctaATG 17C Same as 2-4 5′- Same as 2-4 Same as 2-4 caatttatttaccgaaataaaaggagggat gcgaATG 35C 5′- 5′- Same as 2-4 Same as 2-4gcgtagtagccggg caatttatttttgtt tgataaggagccgt cacccaaggagtatt aacATGgctaATG 36C 5′- 5′- Same as 2-4 Same as 2-4 gcgtagtagccgggcaatttatttaccga tgataaggagccgt aataaaaggagggat aacATG gcgaATG 36C-8 5′-5′- Same as 2-4 5′- gcgtagtagccggg caatttatttaccga ggaagaattaaggtgataaggagccgt aataaaaggagggat gggacaaggggga aacATG gcgaATG ataATG 72B5′- 5′- 5′- 5′- gcgtagtagccggg caatttatttaccga TCTTCCCACAACAGAAATTTCATACC tgataaggagccgt aataaaaggagggat CTGGCGGACTCCA ACAGGCGAAGGAGaacATG gcgaATG TCATG GAAAAACCATG 105F 5′- 5′- 5′- 5′- gcgtagtagccgggcaatttatttttgtt TCTTCCCACAACA GAAATTTCATACC tgataaggagccgtcacccaaggagtatt CTGGCGGACTCCA ACAGGCGAAGGAG aacATG gctaATG TCATGGAAAAACCATG

The expression performance of the RBS-optimized H. elongata ACCases wasevaluated by 3-HP production in a 96-well format, each in triplicatewells, and the averaged results shown in Table 3. Specific 3HPproduction is shown as g/L per OD₆₀₀. As may be seen in Table 3,enhancing the efficiency of the RBS in strains B2, 35C, and 72 B clearlyresulted in increased malonyl-CoA production leading to increased 3-HPproduction. It is evident from these results that combinations ofenhanced RBS's before each of the individual genes accA, accB, accC, andaccD may result in strains with even higher ACCase expression andactivity.

TABLE 3 Improvement in 3-HP production by RBS-optimized expression of H.elongata ACCase subunits. H. elongata ACCase 3HP expression plasmid (g/l· OD) Parent 2-4 0.06 B2 0.81 13A 0.01 14C 0.54 15C 0.14 17C 0.08 35C0.68 36C 0.31 36C-8 0.31 72B 0.57 105F 0.19

Example 4: Coordinated Expression by Subunit Fusions

In nature ACCase subunit genes from prokaryotes such as E. coli and H.elongata have been shown to have a quaternary structure:accA₂:accD₂:accB₄:accC₂. However, the intrinsic levels of the ACCasesubunit genes are too low for optimal production. Therefore, for optimalproduction it is ideal to have overexpression to be coordinated in asimilar manner.

Expression of the genes encoding each ACCase subunit is regulated attranscriptional and translational levels. Coordinated overexpression ofthe ACCase subunit genes, accA, accB, accC, accD should give betterACCase activity. Examples of fusions of accC-B proteins exist inbacteria. It is hypothesized that coordinated overexpression may beachieved by fusion of subunit genes should ensures equimolar expressionof the subunit genes at the optimal time.

The following ACCase subunit gene fusion were constructed and theconstructs overexpressed in E. coli: (A) Control ABCD, (B) fusion ofaccC-B (SEQ ID NO. s 9, 10) subunit genes as seen in bacteria, (C)fusion of accD-A subunit genes using a flexible 15-amino acid linker(Linker sequence LSGGGGSGGGGSGGGGSGGGGSAAA; SEQ ID NO. s 11, 12) asdepicted in FIG. 5.

The performance of the ACC fusions were tested for their ACCase activityand for various 3-HP production metrics in Table 4. ACCase activity wasdetermined in cell lysates using an assay for malonyl-CoA production asdescribed in [Kroeger, Zarzycki, and Fuchs, Analytical Biochem.411:100-105, 2011]. Production of 3-HP was determined in cellsco-transformed with a plasmid bearing the genes encoding the malonyl-CoAreductase from S. tokadaii and E. coli ydfG providing a 3-HPdehydrogenase to complete the metabolic pathway from malonyl-CoA to 3HP.These results show that the strain with the fused accDA genes had higheraverage specific productivity of 3-HP compared to the parental strain inwhich the overexpressed ACCase is not fused. FIG. 6 shows that thebenefit of the accDA fusion were also manifested in 3-HP production infermentors with environmental controls of nutrient feed, pH, aeration,and temperature.

TABLE 4 ACC Fusions and ACCase activity Avg specific Avg specific ACCaseprodn rate prodn rate specific Strain (g/gDCW · h) (g/gDCW · hr)activity at designation Plasmid at TS + 6 at TS + 20 TS + 6 (U/mg)BX3_783 Parent (unfused 0.160 0.146 0.057 ACCase) BX3_829 No ACC 0.0690.062 0.000 BX3_837 EC ACC DA 0.209 0.201 0.054 fusion

Example 5: 3-HP Exporter

Growth inhibition has been demonstrated for E. coli strains grown in thepresence of 3-HP at levels as low as 20 g/L. To produce high titers of3-HP the production host is required to balance production withovercoming inhibition. A known chemical exporter from E. coli that hasbeen previously characterized for homoserine transport, rhtA, wasevaluated for increased production of 3-HP. A mutant version of theexporter, rhtA(P2S) (SEQ ID NO. 30 nucleic acid, SEQ ID NO. 31 protein)was synthesized behind the PtpiA promoter and inserted into thepTRC-PyibD-MCR plasmid behind a terminator using the Gibson Assemply kit(NEB) according to manufacturer's instructions. The effects ofoverexpression of rhtA were evaluated in 1 L fermentation compared tothe control plasmid without rhtA. As shown in FIG. 7, overexpression ofrhtA resulted in a significant improvement in 3HP titer compared to thecontrol production strain. Construction of plasmids expressing anotherputative transporter, ydcO (SEQ ID NO. 32 nucleic acid, SEQ ID NO. 33protein) is carried out in the same manner.

Example 6 Bicarbonate Importer (Prophetic)

Increased import of bicarbonate to increase availability of bicarbonatefor the ACCase reaction will increase production of malonyl-CoA andhence products derived metabolically from malonyl-CoA, such as 3-HP. Thegene encoding the bicA bicarbonate transporter (SEQ ID NO. 13) ofSynechococcus sp. was synthesized using codons optimized for expressionin E. coli (SEQ ID NO. 14) and expressed using the E. coli tal promoterin a strain cotransformed with plasmids encoding ACCase and MCRfunctions. Production of 3-HP by this strain is compared to thatachieved by a control strain without overexpressed bicA.

1.-39. (canceled)
 40. A genetically modified organism capable ofproducing a chemical product of interest, wherein the geneticmodification includes introduction of nucleic acid sequences coding forpolynucleotides encoding salt-tolerant enzymes.
 41. The geneticallymodified organism of claim 40, wherein said polynucleotides encode forone or more enzymes from salt-tolerant organisms that are homologs of akey enzyme selected from the group consisting of: Sucrose-6-phosphatehydrolase (cscA from E. coli), glucose-6-phosphate isomerase (pgi fromE. coli), fructokinase (cscK from E. coli), fructose-1,6-bisphosphatase(yggF from E. coli), fructose 1,6-bisphosphatase (ybhA from E. coli),fructose 1,6-bisphosphatase II (glpX from E. coli),fructose-1,6-bisphosphatase monomer (fbp from E. coli),6-phosphofructokinase-1 monomer (pfkA from E. coli),6-phosphofructokinase-2 monomer (pfkB from E. coli), fructosebisphosphate aldolase monomer (fbaB from E. coli), fructose bisphosphatealdolase monomer (fbaA from E. coli), triose phosphate isomerase monomer(tpiA), glyceraldehyde 3-phosphate dehydrogenase-A monomer (gapA from E.coli), phosphoglycerate kinase (pgk),2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmM fromE. coli), 2,3-bisphosphoglycerate-dependent or tdcE (from E. coli),phosphoglycerate mutase (gpmA), enolase (eno from E. coli),phosphoenolpyruvate carboxylase (ppc from E. coli), malate dehydrogenase(mdh), fumarase A (fum from E. coli), fumarase B (fumB), fumarase C(fumC from E. coli), phosphoenolpyruvate synthetase (ppsA from E. coli),pyruvate kinase I monomer (pykF from E. coli), pyruvate kinase IImonomer (pykA from E. coli), fumarate reductase (frdABCD from E. coli),lipoamide dehydrogenase (lpd from E. coli), pyruvate dehydrogenase (aceEfrom E. coli), pyruvate dehydrogenase (aceF from E. coli), pyruvateformate-lyase (pflB from E. coli), acetyl-CoA carboxylase (accABCD fromE. coli), malonyl CoA reductase (mcr), 3HP dehydrogenase (mmsB, NDSD, orydfG), and malonate semialdehyde reductase (nemA, rutE from E. coli).42. The genetically modified organism of claim 41, wherein said homologsinclude enzymes that have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%overall amino acid or nucleotide identity to said key enzyme.
 43. Thegenetically modified organism of claim 41, wherein said homologs includeenzymes that have at least at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%,90%,89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overallamino acid or nucleotide identity to the essential protein functiondomains of said key enzyme.
 44. The genetically modified organism ofclaim 41, wherein said homologs include enzymes that have at least 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%,89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide identity toessential binding amino acids within an essential protein functiondomain of said key enzyme.
 45. (canceled)
 46. The genetically modifiedorganism of claim 41, wherein said salt-tolerant organism is selectedfrom the group consisting of Halomonas elongata, Salinibacter rubur, andHalobacterium species (Archaea). 47-53. (canceled)
 54. A geneticallymodified organism capable of producing a chemical product of interest,wherein the genetic modification includes introduction of nucleic acidsequences coding for polynucleotides encoding a gene that (1)facilitates importation from an extracellular media to within a cell ofa reactant, precursor, or metabolite used in the organism's productionpathway for producing the chemical product of interest; or (2) inhibitsexportation of said reactant, precursor, or metabolite from within thecell membrane to the media.
 55. The genetically modified organism ofclaim 54, wherein the reactant, precursor, or metabolite is carbondioxide or bicarbonate.
 56. The genetically modified organism of claim55, wherein said gene is selected from the group consisting of bicA fromSynechococcus species, ychM gene product of E. coli, and yidE geneproduct of E. coli.
 57. A method of producing 3-HP using the geneticallymodified organism of claim 40, wherein the organism's reaction pathwayconverts sugar to malonyl-CoA through a series of intermediate steps,converts malonyl-CoA to malonate semialdehyde, and converts malonatesemialdehyde to 3-HP. 58-141. (canceled)
 142. A method of producing 3-HPusing the genetically modified organism of claim 54, wherein theorganism's reaction pathway converts sugar to malonyl-CoA through aseries of intermediate steps, converts malonyl-CoA to malonatesemialdehyde, and converts malonate semialdehyde to 3-HP.
 143. A methodof producing 3-HP using the genetically modified organism of claim 55,wherein the organism's reaction pathway converts sugar to malonyl-CoAthrough a series of intermediate steps, converts malonyl-CoA to malonatesemialdehyde, and converts malonate semialdehyde to 3-HP.